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Response by the author.
Proc Natl Acad Sci USA. 1996; 93(25): 14315.It would be too easy to talk past each other by reference to equations that are nothing more than an expression of the concepts that one holds as valid before writing any equations. It seems more profitable to refer to these concepts directly and fix attention on the differences between our respective beliefs.
In my view the point at issue is as follows: Must one distinguish from the start between the changes in entropy of system and surroundings (total entropy) and the change in entropy within the chemical system (intrinsic entropy), or is this distinction unnecessary? As I say in my note, Planck distinguished between them, and I have followed his views. Gibbs considered only the total entropy increase and for that purpose assumed that “the reagents are enclosed in a rigid and fixed envelope which is impermeable to and unalterable by any of the substances enclosed, and perfectly non conducting ... [truncated at 150 words]
Gratton E, Mantulin WW, Weber G, Royer CA, Jameson DM, Reininger R, Hansen RWC.
Fluorescence dynamics of biological systems using synchrotron radiation.
Synchrotron Radiation Instrumentation, Argonne National Laboratory, Oct 18-20, 1995.
Rev Sci Instrum. 1996; 67(9): 3363.A beamline for time-resolved fluorescence spectroscopy of biological systems is under construction at the Synchrotron Radiation Center. The fluorometer, operating in the frequency domain, will take advantage of the time structure of the synchrotron radiation light pulses to determine fluorescence lifetimes. Using frequency-domain techniques, the instrument can achieve an ultimate time resolution on the order of picoseconds. Preliminary experiments have shown that reducing the intensity of one of the fifteen electron bunches in the storage ring allows measurement of harmonic frequencies equivalent to the single-bunch mode. This mode of operation of the synchrotron significantly extends the range of lifetimes that can be measured. The wavelength range (encompassing the visible and ultraviolet), the range of measurable lifetimes, and the stability and reproducibility of the storage ring pulses should make this beamline a versatile tool for the investigation of the complex fluorescence decay of biological systems.
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Resolution of the ambiguity of van't Hoff plots by the effect of pressure on the equilibrium.
High-pressure Effects in Molecular Biophysics and Enzymology. By JL Markley, DB Northrop, and CA Royer (Editors). Oxford University Press, 1996. ISBN: 9780195097221
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Persistent confusion of total entropy and chemical system entropy in chemical thermodynamics.
Proc Natl Acad Sci USA. 1996; 93(15): 7452-7453. PMCID: PMC38764The change in free energy with temperature at constant pressure of a chemical reaction is determined by the sum (dS) of changes in entropy of the system of reagents, dS(i), and the additional entropy change of the surroundings, dS(H), that results from the enthalpy change, dH. A faulty identification of the total entropy change on reaction with dS(i) has been responsible for the attribution of general validity to the expressions (dΔG/dT)p = -ΔS(i) and d(ΔG/T)/d(1/T) = ΔH, which are found in most textbooks and in innumerable papers.
Ehrhardt MR, Erijman L, Weber G, Wand AJ.
Molecular recognition by calmodulin: pressure-induced reorganization of a novel calmodulin-peptide complex.
Biochemistry. 1996; 35(5): 1599-1605.The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by fluorescence spectroscopy. The 1:1 complex (12 microM) formed between apoCaM and the Neurop peptide is extremely sensitive to salt and is half dissociated in less than 0.1 M KCl, suggesting that electrostatic interactions contribute strongly to complex formation. Ion pair interactions are frequently sensitive to high hydrostatic pressure due to electrostriction effects in the solvated ion state. Application of high pressure to the apoCaM.Neurop complex causes a red shift of the Neurop tryptophan emission center of mass and a reduced residual anisotropy but with insignificant reduction in quantum yield. The transition is smooth, reversible, and apparently two-state with a midpoint pressure of approximately 0.8 kbar. The residual anisotropy, quantum yield, and center of mass of the emission spectrum are consistent with the movement of the tryptophan ... [truncated at 150 words]
Weber G, da Poian AT, Silva JL.
Concentration dependence of the subunit association of oligomers and viruses and the modification of the latter by urea binding.
Biophys J. 1996; 70(1): 167-173. PMCID: PMC1224917A theoretical model is presented that accounts for the facilitation of the pressure dissociation of R17 phage, and for the partial restoration of the concentration dependence of the dissociation, by the presence of subdenaturing concentrations of urea. As an indifferent osmolyte urea should promote the stability of the protein aggregates under pressure, and the decrease in pressure stability with urea concentration demonstrates that such indirect solvent effects are not significant for this case, and that the progressive destabilization is the result of direct protein-urea interactions. By acting as a "homogenizer" of the properties of the phage particles, urea addition converts the pressure- induced deterministic dissociation of the phage into a limited stochastic equilibrium. The model establishes the origin of the uniform progression from the stochastic equilibrium of dimers, to the temperature-dependent and partially concentration-dependent association of tetramers, to the fully deterministic equilibrium observed in many multimers and in the virus ... [truncated at 150 words]
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Pressure-induced dissociation and denaturation of allophycocyanin at subzero temperatures.
J Biol Chem. 1995; 270(48): 28759-28766.The thermodynamics of assembly of the allophycocyanin hexamer was examined employing hydrostatic pressures in the range of 1 bar to 2.4 kbar and temperatures of 20 to -12 degrees C, the latter made possible by the decrease of the freezing point of water under pressure. The existence of two processes, dissociation of the hexamer into dimers, (alpha beta)3->3 (alpha beta), and dissociation of the alpha beta dimers into monomers, (alpha beta)->alpha + beta have been recognized previously by changes in the absorbance and fluorescence of the tetrapyrrolic chromophores owing to added ligands. The same changes are observed in the absence of ligands at pressures of under 2.4 kbar and temperatures down to -12 degrees C. On decompression from 2.4 kbar at 0 degrees C, appreciable hysteresis and a persistent loss of 50% in the absorbance at 653 nm is observed. It results from the conformational drift of the isolated subunits ... [truncated at 150 words]
Reininger R, Hansen RWC, Gratton E, Mantulin WW, Weber G, Royer CA.
Studies of fluorescence dynamics in biological systems using the pulsed structure of the SRC.
APS X-ray Centennial Symposium and 7th Users Meeting for the APS, Argonne, IL, 16-20 Oct 1995.
A beamline for time-resolved fluorescence spectroscopy of biological systems is under construction at the Synchrotron Radiation Center. The fluorometer, operating in the frequency domain, will take advantage of the time structure of the synchrotron-radiation light pulses to determine fluorescence lifetimes. Using autocorrelation techniques, the instrument can achieve an ultimate time resolution on the order of picoseconds. Preliminary experiments have shown that reducing the intensity of one of the fifteen electron bunches in the storage ring allows measurement of harmonic frequencies equivalent to the single bunch mode. This significantly extends the range of lifetimes that can be measured. The wavelength range (down to 200 nm), the range of measurable lifetimes, and the stability and reproducibility of the storage ring pulses should make this beamline a versatile tool for the investigation of the complex fluorescence decay of biological systems.
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Answer to the comments on G. Weber's "van't Hoff revisited: enthalpy of association of protein subunits" by A. Holtzer and by R. Ragone, G. Colonna, and L. Ambrosone.
J Phys Chem. 1995; 99(34): 13051-13051.
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Fluorescence in biophysics: accomplishments and deficiencies.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
The technological advances in the measurements of the fluorescence parameters: spectra, lifetime, yield and polarization have all made impressive advances since the decade of 1920-1930 when techniques were first developed for their measurement. While Gaviola (1926) was able to measure fluorescein lifetime with an accuracy of ±0.5ns, today it is possible to reach into the picosecond and even the femtosecond orders of magnitude. Very weak emissions and heterogeneity of emission, which are encountered in many biological and medical applications presents nowadays few problems as regards detection. Technical improvements have made possible the application of fluorescence to several forms of microscopy with spatial resolutions of the order of one wavelength, and even better resolutions may become possible in the future. On the other hand the physical interpretation of the fluorescence observations has not undergone many changes in the same period. Competitive transitions that determine the fluorescence yield and environmental effects ... [truncated at 150 words]
Jurkiewicz E, Villas-Boas M, Silva JL, Weber G, Hunsmann G, Clegg RM.
Inactivation of simian immunodeficiency virus by hydrostatic pressure.
Proc Natl Acad Sci USA. 1995; 92(15): 6935-7. PMCID: PMC41445The inactivation of the simian immunodeficiency viruses SIVmac251 and SIVagm by pressures of 150 and 250 MPa was determined. The extent of inactivation depended on the time that the virus was subjected to compression as well as the level of the pressure and at 150 Mpa reached 5 log10 dilution units after approximately 10 hr. The inactivations, which were uniformly carried out at room temperature, were independent of the concentration of the virus. Possible applications of pressure inactivation for molecular biological and clinical use are discussed.
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Van't Hoff revisited: enthalpy of association of protein subunits.
J Phys Chem. 1995; 99(3): 1052-1059.The amounts of heat absorbed from and released to the environment in a chemical reaction are not
experimentally separable. In consequence, entropy and enthalpy of reaction can only be computed with the help of specific hypotheses that relate them. We adopt here a previously described model in which enthalpy and entropy changes on reaction are determined by the thermally dependent probability of bond breakage, and we derive a simple function of that probability that gives the number of Boltzmann complexions associated to a set of bonds of arbitrary strength. The general relation proposed by Born for the dependence of bond energy upon distance is used to demonstrate that increase in pressure from atmospheric to 2.5 kbar produces much larger dissociating effects than increase in temperature from 0 to 40 °C. In protein oligomers the energies of protein-protein bonds are of the same order as the thermal energy, and both enthalpy and ... [truncated at 150 words]
Erijman L, Lorimer GH, Weber G.
Plurality of protein conformations of ribulose-1,5-bisphosphate carboxylase/oxygenase monomers probed by high pressure electrophoresis.
J Biol Chem. 1993; 268(34): 25914-25919.We used hydrostatic pressure in the range of 1 to 2 kbar, coupled with polyacrylamide gel electrophoresis, to investigate the properties of monomers of dimeric ribulose bisphosphate carboxylase/oxygenase. At temperatures below -5 degrees C or pressures above 1.5 kbar, only a diffuse band with low electrophoretic mobility was observed, which is assigned to a denatured monomer. In gels run at 1.0 kbar and temperatures above 0 degree C, both the wild type and a mutant in which a positively charged Lys at the dimer interface is replaced by a negatively charged glutamic acid displayed several discrete bands with retardation coefficients larger than that of the dimer. Cross-linking due to oxidation of cysteines was not the reason for the multiplicity of bands, which in addition were independent on the length of the electrophoretic run in the range of 1-3 h. Binding of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid to the pressure dissociated monomers stabilized the ... [truncated at 150 words]
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Pressure stability of proteins.
Annu Rev Phys Chem. 1993; 44: 89-113.The stability of proteins toward temperature has been explored in much detail since the time that proteins were characterized as chemicals of constant composition, but the study of the effects of pressure upon proteins is much :more recent and has been much less frequent than that of temperature. However, there is every reason to expect that the effects of pressure would be more amenable to interpretation than those of temperature: An increase in temperature changes both the energy content and the volume of the system, and because proteins are flexible polymers that maintain secondary, tertiary, and quatenary structure by bonds of strengths not much larger than the thermal energy, the internal interactions of the protein are changed by temperature in ways that cannot be easily foreseen. On the other hand, application of pressure affects internal interactions exclusively by the changes in the distances (volumes) of the components, whereas the total ... [truncated at 150 words]
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Bioenergetics at the end of the XX century: a personal perspective.
Braz J Med Biol Res. 1993; 26(4): 425-437.Three problems of energetics brought to light by recent studies of protein associations are discussed: 1. The anomalous concentration dependence of well-characterized protein dimers demonstrates the shortcomings of the principle of detailed balance as applied to specific macromolecular associations. 2. A rapidly established pressure-induced equilibrium between tetramers and monomers is followed by a much slower exchange of the monomers with the remaining tetramers, indicating the existence of a heterogeneous macromolecular population as regards the free energy of dissociation. The fractions of this population interchange with each other very slowly in comparison with the tetramer-monomer equilibria within each of them...
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Thermodynamics of the association and the pressure dissociation of oligomeric proteins.
J Phys Chem. 1993; 97(27): 7108-7115.A relation giving the distribution of the energies and the entropy of a set of identical bonds as a function of a suitably defined bond strength E is derived. The resulting normal distributions have energies that increase monotonically with E and entropies with a maximum at E/RT=ln 2 and almost negligible values at E/RT>5-7. Association of two protein subunits involves conversion of two sets of protein-water bonds (P-W) into separate water-water (W-W) and protein-protein (P-P) sets, and the constant W-W bond strength permits the determination of the P-P and P-W bond strengths from the experimental enthalpies and entropies of subunit association...
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Physical heterogeneity of muscle glycogen phosphorylase revealed by hydrostatic pressure dissociation.
Biochemistry. 1993; 32(24): 6295-6301.Four independent methods that employ fluorescence spectroscopy show that the tetramer of glycogen phosphorylase A (GPA) from rabbit muscle is reversibly dissociated into monomers by hydrostatic pressures under 2.5 kbar, if aggregation of the monomers is prevented by the addition of 8% glycerol. The free energy of association at 20 degrees C (-32 kcal mol-1) depends upon a large entropy increase (T delta S = +65 kcal mol-1) that counteracts an unfavorable enthalpy of association of +33 kcal mol-1. The association volumes calculated from the pressure dependence of the dissociation are nearly 4-fold smaller than those calculated from the shift in dissociation pressure with concentration. The dimer obtained by dilution of GPA at atmospheric pressure differs from the hypothesized dimer intermediate in the pressure dissociation by the much larger monomer affinity of the former. Like other tetramers, GPA shows hysteresis of the pressure profile upon decompression and conformational drift of ... [truncated at 150 words]
da Poian AT, de Oliveira AC, Gaspar LP, Weber G.
Reversible pressure dissociation of R17 bacteriophage. The physical individuality of virus particles.
J Mol Biol. 1993; 231(4): 999-1008.In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage. In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles. At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation. At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure. In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea. In ... [truncated at 150 words]
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Pressure dissociation of the smaller oligomers: dimers and tetramers.
NATO Advanced Study Institute. Aquafredda di Maratea, Italy. September 20-October 3, 1992.
High Pressure Chemistry, Biochemistry and Materials Sciences (NATO Science Series C, Vol. 401). By R Winter and J Jonas (Editors). Kluwer Academic Press, pp. 471-487, 1993. ISBN: 9780792322900The dissociating effects of pressure upon the oligomeric proteins have been qualitatively observed in large and complex aggregates and also in homogeneous dimers and tetramers, but only the latter permit, on account of their simple stoichiometry, a determination of the thermodynamic parameters. Dieters behave under pressure as simple homogeneous systems with a well defined association volume and unique free energy of association while tetramers appear to be heterogeneous populations as regards these two properties. Interconversion of the members of the tetramer population is temperature-dependent with an energy of activation of 20 kcal mol-1. Tetramers and most dimers show a loss in the free energy of association, and time-dependent recovery when the subunits are separated. This is attributed to a conformational drift, that occurs when intersubunit contacts are replaced by water-subunit contacts. The conformational drift on dissociation results in a change in equilibrium constant with the degree of dissociation in certain ... [truncated at 150 words]
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Relations of bond energies and entropies with volume, pressure and temperature in protein aggregates.
NATO Advanced Study Institute. Aquafredda di Maratea, Italy. September 20-October 3, 1992.
High Pressure Chemistry, Biochemistry and Materials Sciences (NATO Science Series C, Vol. 401). By R Winter and J Jonas (Editors). Kluwer Academic Press, pp. 489-509, 1993. ISBN: 9780792322900A procedure is described to compute the average energies of the protein-protein and protein-water bonds that determine the equilibrium between a protein aggregate and its subunits, starting from the standard enthalpy and entropy changes of subunit association. In four dimers, one trimer and two tetramers the data are unequivocal in deciding that, in contrast to previously held notions, the entropy change that drives the association arises from the conversion of the strong protein-water bonds into much weaker protein-protein bonds. The effects of temperature and pressure on the energy of the bonds may be computed by introducing the intermolecular potentials typical of apolar and dipolar interactions, and by specification of the proportion of each type that charcaterizes the protein-protein and protein-water bonds. The dissociating effect of pressure is shown to depend upon the differential compressibility of the bonds which preferentially destabilizes the more apolar protein-protein bonds.
Erijman L, Lorimer GH, Weber G.
Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase.
Biochemistry. 1993; 32(19): 5187-95.Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15 degrees C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (T delta S) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as ... [truncated at 150 words]
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Use of sensitized fluorescence for the study of the exchange of subunits in protein aggregates.
Photochem Photobiol. 1993; 57(3): 411-415.The exchange of subunits between oligomer protein particles depends upon a cycle of dissociations and associations. To examine the dynamics of these cycles we have employed two methods based on the transfer of excitation energy between fluorochromes attached to different subunits of protein oligomers, at various temperatures and pressures. In the heterotransfer method, identical solutions independently labeled with two different fluorophores, donor D and acceptor A, are mixed. The fluorescence spectrum permits the determination of the subunit exchange by the increase in A and decrease in D fluorescence as mixed AD oligomers are formed. In the homotransfer method the aggregates are labeled with fluorescein to the extent that, ideally, each subunit carries a fluorophore. The emission is strongly depolarized because sufficiently often it takes place after a transfer to a fluorophore oriented differently from the one originally excited. Both dissociation and subunit exchange with unlabeled material result in an increase ... [truncated at 150 words]
Foguel D, Chaloub RM, Silva JL, Crofts AR, Weber G.
Pressure and low temperature effects on the fluorescence emission spectra and lifetimes of the photosynthetic components of cyanobacteria.
Biophys J. 1992; 63(6): 1613-1622. PMCID: PMC1262278The effects of hydrostatic pressure on the excited state reactions of the photosynthetic system of cyanobacteria were studied with the use of stationary and dynamic fluorescence spectroscopy. When the cells were excited with blue light (442 nm), hydrostatic pressure promoted a large increase in the fluorescence emission of the phycobilisomes (PBS). When PBS were excited at 565 nm, the shoulder originating from photosystem II (PSII) emission (F685) disappeared under 2.4 kbar compression, suggesting suppression of the energy transfer from PBS to PSII. At atmospheric pressure, the excited state decay was complex due to energy transfer processes, and the best fit to the data consisted of a broad Lorentzian distribution of short lifetimes. At 2.4 kbar, the decay data changed to a narrower distribution of longer lifetimes, confirming the pressure-induced suppression of the energy transfer between the PBS and PSII. When the cells were excited with blue light, the decay at ... [truncated at 150 words]
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Protein Interactions.
Protein Interactions. Chapman & Hall, New York, 1992. ISBN: 9780412030314This book presents a detailed examination of the physical properties of proteins and their associations with solvents, ligands, and each other. Incorporating observations from his classic work on the thermodynamics of protein-protein and protein-ligand interactions, the internationally recognized author offers a complete conceptual and quantitative description of proteins and their associations. The book begins with an overview of basic thermodynamic principles and goes on to describe the covalent and noncovalent binding of single and multiple ligands and their effects on protein-protein associations. Discussions of protein-solvent interactions address issues including protein folding, membrane associations, and protein dynamics. Attention is given to the effect of temperature and pressure on protein structure, oligomerization, and ligand binding, as well as to ideas about the basis of biological specificity. Although experimental work is discussed throughout the text, the book is not technique oriented. Its theoretical framework is clear, general and easily understandable, making this an ... [truncated at 150 words]
Silva JL, Luan P, Glaser M, Voss EW Jr, Weber G.
Effects of hydrostatic pressure on a membrane-enveloped virus: high immunogenicity of the pressure-inactivated virus.
J Virol. 1992; 66(4): 2111-2117. PMCID: PMC289002A new approach to the preparation of antiviral vaccines relying on the inactivation of the virus particle by hydrostatic pressure is described. The enveloped virus vesicular stomatitis virus was utilized as a model; a pressure of 260 MPa applied for 12 h reduced infectivity by a factor of 10(4), and the antibodies against pressurized material were as effective as those against the intact virus when measured by their neutralization titer. Fluorescence measurements indicate that application of pressure results in perturbations of the particle interactions that permit binding of specific molecular probes. Electron microscopy showed that the membrane of the pressurized virus was partially preserved, presenting the spike pattern of the membrane G protein. Unlike the icosahedral viruses, dissociation into smaller particles was not observed, but a constant change in the morphology was the presence of a bulge in the surface of the pressurized virus, indicating a displacement of the capsid ... [truncated at 150 words]
Erijman L, Lorimer GH, Weber G.
Carbamylation protecis dimeric rubisco from inactivation after dissociation by hydrostatic pressure.
36th Annual Meeting of the Biophysical Society, Houston, Texas, 9-13 February 1992.
Biophys J. 1992; 61(2 Pt 2): A475, 2739.Hydrostatic pressure coupled with fluorescence and absorption spectroscopy has been used to investigate protein subunit interactions in dimeric ribulose biphosphate carboxylase from Rhodospirllum rubrum. A two fold decrease in the rotational relaxation time reveals the dissociation of the AEDANS labeled dimer. The free energy of association of the monomers at atmospheric pressure is -10.5 kcal mol-I at 20C, with a volume change on association of 100-120 ml mol-1. The conformation and pressure stability of the native and labeled dimer are sensitive to the presence of glycerol and to the activation of the enzyme. Significant differences in absorption and binding of bis(anmlinonaphtalep,e- sulfonate) (BIS-ANS) occur depending on the presence of Mg' and NaHCO3. The carbamylated protein is fully active after a cycle of compression-decompression. On the other hand, no spontaneous or chaperonin assisted reconstitution of activity is detected in samples subjected to pressure induced dissociation in non activated enzyme. In the ... [truncated at 150 words]
da Poian AT, de Oliveira AC, Gaspar LP, Weber G, Silva JL.
Pressure dissociation of RNA phages. The urea-induced transition from deterministic to stochastic dissociation.
36th Annual Meeting of the Biophysical Society, Houston, Texas, 9-13 February 1992.
Biophys J. 1992; 61(2 Pt 2): A472, 2722.R17 bacteriophage capsid is composed by 180 copies of a coat protein and one copy of the A protein, which is necessary to virus infection. Hydrostatic pressure was utilized to promote dissociation of R17 phage and of its constitutive unit, the coat protein dimer. The effects of pressure were monitored by changes in fluorescence emission, hydrodynamic properties and viability assays. Pressures up to 2.5 kbar promoted only 10 % of dissociation of the whole phage, whereas the coat protein dimer was fully dissociated in this pressure range. This result suggests that binding to RNA confers stability to the subunit interaction. The phage was also very stable to urea dissociation and denaturation, since only urea concentrations above 5.0 M affected its structure and viability. Complete dissociation of the phage particles could be obtained by the combined use of pressure and urea. The pressure dissociation of R17 virus in presence of 2.5 ... [truncated at 150 words]
Foguel D, Chaloub RM, Silva JL, Crofts AR, Weber G.
Pressure and low temperature studies on the dissociation of phycobilisomes (PBS) and allophycocyanin (APC) from blue green algae.
36th Annual Meeting of the Biophysical Society, Houston, Texas, 9-13 February 1992.
Biophys J. 1992; 61(2 Pt 2): A103, 599.Phycobiliaomes are high-molecular weight aggregates composed of phycobiliproteins which act as the light-harvesting pigments in blue-green and red algae. We have utilized hydrostatic pressure and low temperature as a tool to promote dissociation of this macromolecular complex and of its trimeric core constituent, APC. Pressures up to 2.4 kbar at room temperature did not promote any considerable alteration in the PBS emission spectra or in the APC absoption spectra. On the other hand, when the titrations of pressure were done at low temperature, or when the temperature was decreased under 2.4 kbar, we observed a noticeable blue shift (670 to 655 nm) in the emission spectra of the PBS, indicating its dissociation. The same pattern was observed for the trimeric protein (APC), in which the decrease in temperature at 2.4 kbar promoted the disappearance of the 653 nm absorption peak related to the trimeric state, suggesting complete dissociation to monomers. ... [truncated at 150 words]
Pin S, Royer CA, Gratton E, Alpert B, Weber G.
Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques.
Biochemistry. 1990; 29(39): 9194-202.The dissociation of the subunits of human adult oxyhemoglobin has been investigated by using steady-state fluorescence anisotropy, multifrequency phase fluorometry, and high hydrostatic pressure. Human hemoglobin obtained by using two purification procedures (bulk preparation by centrifugation or further fractionation using anion-exchange chromatography) was labeled with an extrinsic fluorescent probe, 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl). The long fluorescence lifetime of this probe allows for the observation of the macromolecular tumbling, and thus provides a method for observing changes in the size of the complex upon subunit dissociation under differing solution conditions of proton and organic phosphate concentration. At pH 7, the dansylated preparations of bulk and fractionated hemoglobin showed a concentration-dependent decrease in the anisotropy which though not identical can only arise from the tetramer to dimer dissociation. We observed primarily the dimer at pH 9 and a small destabilization of the tetramer in the presence of saturating inositol hexaphosphate (IHP). High-pressure experiments ... [truncated at 150 words]
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Whither Biophysics?.
Annu Rev Biophys Biophys Chem. 1990; 19: 1-6.Excerpt: When I arrived in Cambridge, England from my native Argentina at the end of 1943, I soon visited the famous Cavendish Laboratory. Just before the war, a small detached building had been constructed to house the cryophysics unit that Pieter Kapitza directed previously to his then recent return to Russia. On the outside wall of the building there was a handsome bas-relief of a crocodile by Eric Gill; Kapitza had thought of it as a symbol of science, which, like the crocodile cannot look backwards. When a scientist with many years of experience is asked for a contribution like the present he usually looks back and reminisces over his years, but, in accordance with my crocodilean nature I have chosen to look into the present and future, which, in science at least, are usually more interesting than the past...
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Local motions of fluorophores.
BioMetals. 1990; 3(2): 127-130.We further describe the general formulation of fluorescence depolarization in which the depolarization results from exchanges between a number of oscillator orientations in thermal equilibrium. Temperature and pressure affect the polarization by changing the relative populations of the allowed orientations as well as the rate of exchanges during the fluorescence lifetime. This treatment satisfactorily describes the limited motions that fluorophores undergo when they are either attached to a macromolecule such as the local tryptophan rotations in proteins, or embedded in a biological membrane.
Foguel D, Silva JL, Chaloub RM, Weber G.
Effects of hydrostatic pressure on the fluorescence properties of cyanobacteria.
34th Annual Meeting of the Biophysical Society, Baltimore, Maryland, February 1990.
Biophys J. 1990; 57(2 Pt 2): 567a, W-Pos592.At room temperature, the fluorescence of cyanobacteria is due mainly to PSII. PSI fluorescence only appears at temperatures close to 4 Kelvin. Hydrostatic pressure provoked a great increase in the three main emission components (PSII, PSI and APCPC). At 2.5 kbar, the accessory pigments emission (Amax- 655 nm) increased 11-fold whereas emission of PSII 0, a 685 nm) only increased 2-fold, suggesting a decrease in the energy transfer from the accessory pigments to clrorophylll a. At one tm. the excited state decay could only be fitted to bimodal distribution of short lifetimes. At 2.5 kbar, the decay was fitted to a single distribution of longer lifetimes confirming the supression of the energy transfer. The emission of PSI als8 became pronounced at 2.5 kbar (20 C) and the intensity (4-730 nm) increased many-fold when the temperature was reduced to -5 C.
Royer CA, Pin S, Weber G, Alpert B, Gratton E.
Subunit interactions and allosteric effects in hemoglobin: high pressure fluorescence studies.
34th Annual Meeting of the Biophysical Society, Baltimore, Maryland, February 1990.
Biophys J. 1990; 57(2 Pt 2): 232a, Tu-Pos45.The dissociation of human hemoglobin labeled with the covalent fluorescence dye, 1-5 dansyl, was studied by monitoring the polarization and lifetime of the dansyl emission as a function of concentration and pressure. The apparent rotational volumes extracted from Ulese data were used to indicate the oligomeric state of the protein. Dissociation profiles were obtained for standard hemo obin as well as for the major component of a further DEAE purification at pH 7 in absence and in presence of the allosteric effector (inositol hexaphosphate) and at H 9. Our results show a destabilization of the 2,2 tetramer at high pH for both preparations and a stabilization of the tetramer at pH 7 for the major component as compared to the standard preparation. High pressure experiments resulted in hemoglobin monomer formation indicating that the al,l interface interactions are weaker than enerallv assumed. (Supported in part by PHS-41-RR03f55 and French Foreign Ministry.)
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From solution spectroscopy to image spectroscopy.
Cell Structure and Function by Microspectrofluorometry (Analytical Cytology Series). By E Kohen and JG Hirschberg (Editors). Academic Press, pp. 71-85, 1989. ISBN: 9780124177604
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Dynamics of oligomeric proteins.
J Mol Liquids. 1989; 42: 255-268.Pressure dissociation of oligomeric proteins is followed by a time-dependent loss in free energy of subunit association. This is revealed by hysteresis in the cycle of compression and decompression and by slow recovery of enzymic activity and spectral properties after decompression. It is attributed to a “conformational drift” of the separated subunits which brings about a heterogeneous population of aggregates after decompression. The often-observed cold inactivation of oligomeric enzymes appears to proceed through a cycle of dissociation, monomer conformational drift and formation of enzymically inactive aggregates, and a cycle gives rise to the dependence of the free energy of association upon the degree of dissociation observed in some dimer proteins. This latter phenomenon is clearly predicted by a stochastic simulation of the concentration dependence of dimer-monomer equilibria with and without drift. By extension from the observed phenomena, and the theory that underlies them, it is concluded that oligomeric aggregates are ... [truncated at 150 words]
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Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase.
Biochemistry. 1989; 28(5): 2144-2153.Pressure dissociation of yeast glyceraldehydephosphate dehydrogenase (GAPDH) was studied by fluorescence spectroscopy. Observations in the range of -5 to 30 degrees C indicate that monomer association into the tetramer proceeds with an enthalpy change of -14 kcal mol-1 and a large increase in entropy which at 25 degrees C amounts to 18 kcal mol-1. The large conformational drift and the low-temperature stability of the tetramer recovered after decompression facilitated a comparison of its properties with those of the native tetramer. Significant differences in absorption and fluorescence-excitation polarization spectra, yield of tryptophan fluorescence, and binding of anilinonaphthalenesulfonate and NADH were observed. At 0 degree C the standard free energies of association of the monomers into the native and drifted tetramers were respectively -32 and -29 kcal mol-1. The volume change upon association measured from the pressure span of the compression curves was 200-230 mL mol-1 but four times as large when ... [truncated at 150 words]
Paladini AA, Silva JL, Weber G.
Slab gel electrophoresis performed at high hydrostatic pressure: a new approach for the study of oligomeric proteins.
International Symposium in Honor of Gregorio Weber's Seventieth Birthday. September 9-12, 1986. Bocca di Magra, Italy.
Fluorescent Biomolecules: Methodologies and Applications. By DM Jameson and GD Reinhart (Editors). Plenum Press, pp. 101-113, 1989. ISBN: 9781468456219In the last few years several studies have demonstrated the reversible dissociation of oligomeric proteins under pressure (for review see: Heremans, 1982; Weber and Drickamer, 1983). The dissociating effects of pressure are generally ascribed to the intersubunit surfaces and the restrictive effect of the covalent bond architecture of the protein. The pressure dissociation has been detected either by indirect or direct methods. Indirect methods, such as hybridization studies (Jaenicke and Koberstein, 1971) and activity measurements (Penniston, 1971; Schade et al., 1980; Seifert et al., 1985) have been employed with some success. These indirect methods are valuable in revealing qualitatively the involvement of protein dissociation in the observed effect of pressure. However, the stoichiometry of dissociation and the thermodynamic parameters (volume change and dissociation constant) cannot be unambiguously determined. Methods which directly reveal the state of association are more suitable for a thermodynamic approach to the pressure dissociation, although they are ... [truncated at 150 words]
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Final words at Bocca di Magra.
International Symposium in Honor of Gregorio Weber's Seventieth Birthday. September 9-12, 1986. Bocca di Magra, Italy.
Fluorescent Biomolecules: Methodologies and Applications. By DM Jameson and GD Reinhart (Editors). Plenum Press, pp. 343-349, 1989. ISBN: 9781468456219As I thought some time ago about what I was going to tell you, I realized that I would be obliged to say about myself more than what I should or what you may wish to hear. I must then ask you to forgive me if I reminisce a little about my initiation in fluorescence which was probably quite close to the initiation of the uses of fluorescence in Biochemistry. I went to Cambridge from the Argentine in 1943 with a British Council Fellowship and spent my first six months at the laboratory of Eric Riddeal learning Surface Chemistry. I convinced myself that it had no future in Biochemistry. You can see how wrong I was; I could have preceded relevant work on membranes by a long period but I really missed my chance. And it was not for lack of hints: At that time, Jim Danieli, in Cambridge itself, ... [truncated at 150 words]
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Perrin revisited: parametric theory of the motional depolarization of fluorescence.
J Phys Chem. 1989; 93(16): 6069-6973.The usual derivations of the depolarization of fluorescence under continuous illumination treat it as a problem of rotational diffusion following the creation of a gradient of oscillator orientations by photoselection. An alternative formulation is presented in which the depolarization results from exchanges between a fixed number of oscillator orientations in thermodynamic equilibrium. In this formulation the parameters that determine the observed stationary polarization are the relative populations of the allowed orientations, the loss of polarization owing to the orientation exchanges, and the rates of reorientation during the fluorescence lifetime. The standard enthalpy and volume changes in the orientational equilibria determine the changes in polarization with temperature and pressure. This treatment predicts temperature dependencies of the polarization like those observed in the local motions of tyrosines and tryptophans in peptides and proteins and of the porphyrin in its specific complexes with apomyoglobin and apohemoglobin.
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Abrupt transitions in physics and biophysics: van der Waals revisited.
Proc Natl Acad Sci USA. 1987; 84(21): 7359-7362. PMCID: PMC299295An iteration procedure that relates pressure and volume changes in the condensation of a gas by means of two independent relations of the volume and the effective pressure predicts abrupt volume transitions similar to those experimentally observed. Similar abrupt transitions are predicted for a molecular association in which the association free energy is dependent upon the extent of reaction. The pressure, or the concentration, at which the abrupt transition occurs depends upon the volume, or the extent of reaction, used to initiate the iteration procedure and selection of the transition that corresponds to the equilibrium requires additional conditions. For the gas condensation, assumption of the symmetry of the effects of the pressure fluctuations at equilibrium gives results that coincide with those obtained by application of the Maxwell rule. From these observations it is concluded that abrupt transitions arise naturally when independent conditions cannot be simultaneously satisfied.
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Fluorescence spectroscopy at high pressure: techniques and results.
Current Perspectives in High Pressure Biology. By HW Jannasch, RE Marquis, and AM Zimmerman (Editors). Academic Press Inc (London), pp. 235-243, 1987. ISBN: 9780123803856
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Biological membrane modeling with a liquid/liquid interface. Probing mobility and environment with total internal reflection excited fluorescence.
Biophys J. 1987; 52(3): 367-379. PMCID: PMC1330001Total internal reflection of exciting light, in combination with fluorescence intensity and polarization measurements, was used to selectively study fluorescent compounds adsorbed to the interface region between two immiscible liquids. A fluorometer was constructed which provided excitation at variable angles of incidence and allowed sensitive detection of polarized fluorescence emitted from the interface. The compound 4,4'-bis-1-phenylamino-8-naphthalenesulfonate (bis-ANS) was examined at a decalin/water interface and was found to possess remarkable affinity for the interface region with the bulk of the adsorbed molecule residing in the decalin phase. The adsorbed fluorophore displayed an apparent hindered rotation in the plane of the interface with a rotational diffusion coefficient 3- to 12-fold lower than that expected for bis-ANS in solution. While other dyes examined were not found to be significantly surface active, the addition of cationic surfactant sufficed to induce adsorption of the anionic fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid. This fluoropore was found to reside in ... [truncated at 150 words]
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Dissociation of oligomeric proteins by hydrostatic pressure.
NATO Advanced Study Institute on Advances in High Pressure Studies of Chemical and Biochemical Systems, Corfu, Greece. September 28-October 11, 1986.
High Pressure Chemistry and Biochemistry (NATO Science Series C, Vol. 197). By R van Eldik and J Jonas (Editors). pp. 401-420, 1987. ISBN: 9789027724571The methods of study of proteins under high pressure, and the effects of pressure upon proteins made up of a single peptide chain are briefly reviewed. The effects upon oligomeric proteins are examined in detail: The existence of time-dependent changes in the conformation of the dissociated subunits, termed a “conformational drift”, is revealed by the temporary decreases in subunit affinity, enzymic activity and changed spectroscopic properties of the aggregates formed after decompression. Its significance in relation to the equilibria established under pressure is discussed and the insuficiency of conventional descriptions of the chemical equilibrium for these cases is noted.
Paladini AA, Silva JL, Weber G.
Slab gel electrophoresis of oligomeric proteins under high hydrostatic pressure. I. Description of the system and demonstration of the pressure dissociation of a dimer.
Anal Biochem. 1987; 161(2): 358-364.A high-pressure bomb was constructed to study the gel electrophoretic behavior of oligomeric proteins under pressure. The apparatus designed by us allows the use of a polyacrylamide slab gel with a capacity of up to 12 wells, therefore permitting the study of several samples in one experiment. The electrophoresis mobility of different single-chain proteins under pressure decreased in the same proportion and the elution pattern was similar to that of the control run at atmospheric pressure. Densitometric analysis of the gel did not show peak spread or asymmetric boundaries, indicating that their conformations were not drastically affected. On the other hand, high-pressure electrophoresis of a dimer, the tryptophan synthase β2 subunit, revealed the appearance of a second peak not present at atmospheric pressure. The mobility of the second peak was higher and its fraction increased by decreasing the protein concentration, indicating that the extra peak was the dissociated monomer. The ... [truncated at 150 words]
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Free energy couplings between ligand binding and subunit associaton in hemoglobin are of first order.
Biochemistry. 1987; 26(1): 331-332.The calculations presented in a recent paper [Johnson, M. L. (1986) Biochemistry 25, 791-797] to the effect that the free energy couplings between oxygen binding and subunit association in hemoglobin A can be of either first or second order are examined. The fitting of the experimental data to a system with second-order couplings carried out by Johnson belongs to a tetramer in which, in contradistinction to hemoglobin A, oxygen binding promotes subunit association.
Royer CA, Weber G, Daly TJ, Matthews KS.
Dissociation of the lactose repressor protein tetramer using high hydrostatic pressure.
Biochemistry. 1986; 25(25): 8308-8315.Dissociation of lac repressor tetramer by high hydrostatic pressures was monitored with intrinsic tryptophan fluorescence. With the assumption of complete dissociation to monomer, tryptophan polarization data gave delta V a approximately 170 mL/mol and the concentration for 50% tetramer dissociation, C1/2, was 3.8 × 10(-8) M. Upon addition of inducer, the calculated delta V a increased to approximately 220 mL/mol and the C1/2 decreased to approximately 1 × 10(-8) M, a free energy difference of approximately 0.7 kcal. These results indicate a modest stabilization of the tetramer by the presence of inducer. Monitoring the average energy of tryptophan emission demonstrated that tetramer dissociation takes place over the same range of pressures as evidenced by the polarization data and IPTG dissociation can be more or less superimposed upon tetramer dissociation depending upon the ligand concentration used. Although the two transitions cannot be separated entirely, the delta V a for the region ... [truncated at 150 words]
Silva JL, Miles EW, Weber G.
Pressure dissociation and conformational drift of the beta dimer of tryptophan synthase.
Biochemistry. 1986; 25(19): 5780-5786.Micromolar solutions of tryptophan synthase beta 2 dimer dissociate into monomers in the pressure range of 800-1600 bars as shown by studies of the spectral shift of the intrinsic fluorescence and of the fluorescence polarization of dansyl conjugates. At 25 degrees C the standard change in volume on dissociation (dV0) of the holoprotein was -162 mL mol-1, and the dissociation constant at 1 bar was K0 = 3.7 × 10(-10) M. Pyridoxal-reduced holoprotein and apoprotein had, within 10%, the same dV0, but K0 was decreased in the reduced protein (6 × 10(-11) M) and increased in the apoprotein (3.6 × 10(-9) M). At 4 degrees C the free energy of association of the holoprotein was reduced by 1.4 kcal mol-1, but dV0 was unchanged. In all the protein forms the decompression curves differed from the respective compression curves, indicating the loss of some free energy of association following separation of ... [truncated at 150 words]
Verjoski-Almeida S, Kurtenbach E, Amorim AF, Weber G.
Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase.
J Biol Chem. 1986; 261(21): 9872-9878.The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It ... [truncated at 150 words]
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Phenomenological description of the association of protein subunits subjected to conformational drift. Effects of dilution and of hydrostatic pressure.
Biochemistry. 1986; 25(12): 3626-3631.The native conformation of oligomers may be expected to undergo reversible changes when they separate upon dissociation of the original aggregate. When these changes are slow in comparison with the time of an association-dissociation (AD) cycle, they give rise to characteristic effects in the dependence of the dissociation: upon dilution, at constant pressure, and upon the applied pressure, at constant concentration. The phenomenological description of these effects is examined by comparing two possible models: The first model assumes a continuous loss in free energy of association with the extent of dissociation; the second supposes the existence of two or more distinct aggregates differing in subunit affinity and present in proportions that vary with the extent of dissociation. The latter model fits better the experimental data available, with regard to both the concentration and the pressure dependence of the association, and gives a particularly simple explanation of the hysteresis phenomena observed ... [truncated at 150 words]
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Conformational drift of dissociated lactate dehydrogenase.
Biochemistry. 1986; 25(12): 3632-3636.Bovine and porcine lactate dehydrogenases, in solutions of 0.1-10 microM at neutral pH, dissociate into monomers upon application of hydrostatic pressures of up to 2 kbar. The dissociation was determined from observations of the polarization of fluorescence under pressure in seeming equilibrium conditions and by occasional hybridization experiments of the H4 and M4 isozymes. Decompression is followed by the rapid association of the monomers into tetramers and by slow, and sometimes incomplete, return of the enzymic activity. The dissociation curves obtained on compression and decompression differ, indicating that association results in partial loss of subunit affinity. These phenomena are attributed to a slow conformational drift that follows the loss of contact of the monomers with each other and to an even slower reversal of the drift that takes place upon reassociation.
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Conformational drift and cryoinactivation of lactate dehydrogenase.
Biochemistry. 1986; 25(12): 3637-3640.Solutions of porcine lactate dehydrogenase of micromolar concentration kept at 4 degrees C for several days lose the greater part of their enzymic activity but recover it when returned to room temperature. The rate of spoiling decreases and the rate of recovery increases with the concentration of the solutions. The decrease in tetramer stability in the cold is shown by experiments of pressure dissociation at various temperatures and confirmed because isozyme hybridization occurs in parallel with the inactivation at low temperature but is absent at room temperature. Cold-inactivated solutions contain tetramers that dissociate much more readily than those of the fully active solutions. It is postulated that cryoinactivation, like pressure inactivation, takes place through a cycle of dissociation, conformational drift [King, L., & Weber, G. (1986) Biochemistry (second paper of three in this issue)] and reassociation into inactive tetramers.
Silva JL, Miles EW, Weber G.
Pressure-induced dissociation and conformational drift of tryptophan synthase ß2 subunit.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 490a, W-Pos137.The conformational drift is the limited change in structure, rather than complete unfolding, that occurs in oligomeric protein subunits after loss of intersubunit contact. Hydrostatic pressure in the range of 1 to 3 kbar provides a unique method of dissociation that does not affect by itself the tertiary structure and is rapidly reversible. The pressure induced dissociation of tryptophan synthase ß2 subunit was followed by the changes in the intrinsic fluorescence. The dissociation curves revealed a volume change (delta V0) of 160 ml/mol and a dissociation constant at atmospheric pressure of 4 . 10- M. The midpoint dissociation pressure was dependent on the protein concentration as expected for a dimer-monomer equilibrium. A large decrease of the total fluorescence intensity of pyridoxal-P was also found upon dissociation. After release of pressure, the dimeric structure was rapidly recovered (less than 5 min.) as judged by return of the original intrinsic fluorescence spectrum ... [truncated at 150 words]
MacGregor RB Jr, Weber G.
Estimation of the polarity of the protein interior by optical spectroscopy.
Nature. 1986; 319: 70-72.Crystallographic studies of myoglobin have shown that the haem pocket is lined with nonpolar amino-acid residues. In order to estimate the true polarity of the interior of proteins, certain studies have used bound fluorophores2-7, the spectroscopic properties of which reflect the polarity of their environment. These studies have most often used l-amino-8-naphthalene sulphonate (ANS) as a probe, but a more suitable probe, in principle, is 6-propionyl 2-(N,N-dimethyl)aminonaphthalene (PRODAN). We have synthesized a molecule with the advantageous spectroscopic properties of PRODAN but with a higher affinity for apomyo-globlin: 2'-(N,N-dimenthyl)amino-6-naphthopyl-4-trans-cyclo-hexanoic acid (DANCA), and report here its use to determine the polarity of the myoglobin haem pocket. Our results show that the pocket is actually a polar environment, and the polarity can be accounted for by peptide amide dipoles.
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Conformational drift of lactate dehydrogenase.
Biophys J. 1986; 49(1): 72-73. PMCID: PMC1329581The loss and recovery of the enzymic activity of lactate dehydrogenase (LDH) subjected to pressure have been studied by Jaenicke and co-workers (1, 2). On the assumption that the fraction of enzyme activity after decompression accurately represents the degree of dissociation of the tetramer into monomers at the incubation pressure, they calculated a standard change in volume on dissociation of -500 ml/mol. They ascribe the slow reactivation at atmospheric pressure to the reassociation of the monomers. We have used fluorescence polarization methods (3) to monitor the degree of dissociation of LDH under pressures of 1 atmosphere to 3 kbar. When degree of dissociation and enzymic activity are separately measured they reveal a more complex situation than that postulated by Jaenicke et al.
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Solution spectroscopy and image spectroscopy.
Applications of fluorescence in the biomedical sciences. By DL Taylor (Editors). Alan R Liss, pp. 601-615, 1986. ISBN: 9780845142103
Hall RD, Valeur B, Weber G.
Polarization of the fluorescence of triphenylene: a planar molecule with three-fold symmetry.
Chem Phys Lett. 1985; 116(2,3): 202-205.Excitation and emission polarization spectra have been obtained for triphenylene in pmpylene glycol at - 70°C. Within experimental error, the excitation polarization spectrum remains constant at p0 = 0.14 from 270 to 350 nm. No red-edge excitation effects have been observed in the emission spectra produced by center-of-spectrum and red-edge excitation.
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Partition of proteins between water and non-polar phases.
NATO Advanced Study Institute on Physical Methods on Biological Membranes and Their Model Systems. Alto Villa Milicia, Italy. September 2-October 2, 1982.
Physical Methods on Biological Membranes and Their Model Systems (NATO ASI Series A: Life Sciences, Vol. 71). By F Conti, WE Blumberg, J de Gier, and F Pocchiari (Editors). Plenum Press, pp. 397-406, 1985. ISBN: 9781468475401The partition of small molecules between water and a non-polar phase follows simple and well defined rules. The most important macroscopic characteristic that determines partition between these phases is their difference in dielectric constant. The high dielectric constant of water favors the separation of charges with formation of independent ions of opposite charge but in media of much lower dielectric constant, separation of charges cannot take place and in consequence the ions present in the water cannot be transferred to the second phase. The high dielectric constant of water results from the permanent dipole moment of the water molecules and the high dipole density. These properties determine the very different molecular interactions prevalent in water and in a typical non-polar medium. On the water side we have the strong attractive forces between permanent dipoles and in a lipid phase the much weaker dispersion forces that result from the attractive effects ... [truncated at 150 words]
Verjoski-Almeida S, Kurtenbach E, Weber G.
Dissociation of solubilized dimeric sarcoplasmic reticulum ATPase induced by pressure.
29th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 24-28 February, 1985.
Biophys J. 1985; 47(2): 455a, W-Pos193.Sarcoplasmic reticulum ATPase was solubilized with nonionic detergent dodecyl octaethylene glycol monoether (C12E8) under conditions which were known to yield dimeric functional ATPase (Silva and Verjovski-Almeida, Biochemistry 22:707, 1983). Polarization of fluorescence of tryptophan residues and of a pyrene butyryl probe covalently linked to the ATPase were measured. Pressures in the range of 1 bar to 2 kbar promoted dissociation of the ATPase dimers. The pressure required for halfdissociation (p 1/2) varied with ATPase protein concentrations in the range of 8 to 20 pg/ml. The volume changes upon dissociation were in the range of -155 ml/mole to -196 ml/mole. An increase in [Ca2] from 50 pM to 5 mM and in pH from 6.8 to 8.5 increased p 1/2 whereas a decrease in pH to 6.0 was of no effect. The activity of the ATPase decreases with increasing pressures in the rang of 1 bar to 0.8 kbar; the lower ... [truncated at 150 words]
Weber G, Scarlata SF, Rholam M.
Thermal coefficient of the frictional resistance to rotation in simple fluorophores determined by fluorescence polarization.
Biochemistry. 1984; 23(26): 6785-6788.Experimental data are presented by employing organic fluorophores, tryptophan and tyrosine among them, that substantiate a logarithmic expansion of the viscosity as a function of temperature for the determination of the thermal coefficient of the viscosity by measurements of the polarization of the emitted fluorescence. The values obtained agree within experimental uncertainties with those determined by flow viscometry, and the agreement extends to a range of glycerol-water mixtures for which the thermal coefficient of the viscosity is an irregular function of the glycerol content. Prodan, a fluorophore that forms strong bonds with the hydroxylic solvents employed, yields values similar to those obtained with perylene, which interacts with solvent by weak dispersion forces alone.
Scarlata SF, Rholam M, Weber G.
Frictional resistance to local rotations of aromatic fluorophores in some small peptides.
Biochemistry. 1984; 23(26): 6789-6792.We have determined, by observations of the polarization of the fluorescence, the thermal coefficient of the frictional resistance to the rotation of the emitting tyrosine or tryptophan residue in the hormones ocytocin and vasopressin and in other small peptides dissolved in 80% glycerol-water over the temperature range of -40 to +20 °C. The plots of the logarithm of the reduced anisotropy [Weber, G., Scarlata, S., & Rholam, M. (1984) Biochemistry (preceding paper in this issue)] vs. temperature consist of two linear regions meeting at a critical temperature, tc, characteristic for each peptide. In the range t < tc the slope corresponds to the thermal coefficient of the viscosity of the pure solvent (approximately 0.07/°C). At t > tc, the slope changes abruptly to a lower value that varies from 0.0325 to 0.060 in the different peptides. The low-temperature slope corresponds to a "solvent-limited regime" and the high-temperature slope to a ... [truncated at 150 words]
Rholam M, Scarlata SF, Weber G.
Frictional resistance to the local rotations of fluorophores in proteins.
Biochemistry. 1984; 23(26): 6793-6796.The fluorescence polarization of solutions of various proteins dissolved in 80% glycerol-water was measured in the temperature range of -40 to +20 °C, and the results were analyzed by the plots of reduced anisotropy vs. temperature described in two previous papers [Weber, G., Rholam, M., & Scarlata, S. (1984) Biochemistry (first paper of three in this issue); Scarlata, S., Rholam, M., & Weber, G. (1984) Biochemistry (second paper of three in this issue)]. The majority of the plots exhibited features similar to those of the peptides: two linear segments joining at a well-defined critical temperature, a common low-temperature slope corresponding to the thermal coefficient of the solvent viscosity, and a characteristic high-temperature slope in the range 0.040-0.015/°C. The plots of lysozyme and diisopropyl fluorophosphonate-chymotrypsin showed three distinct linear regions. It is concluded that the thermal coefficient of the frictional resistance to rotations in proteins is determined, like in peptides, by ... [truncated at 150 words]
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Order of free-energy couplings between ligand binding and protein subunit association in hemoglobin.
Proc Natl Acad Sci USA. 1984; 81(22): 7098-7102. PMCID: PMC392084The number of protein subunits that must be liganded to effect changes in subunit interaction may be characterized by defining an order for the free energy couplings between these two processes. From available data on the chemical equilibrium of stripped hemoglobin A with oxygen, I show that couplings are unequivocally of first order. The two-state model of cooperative binding is shown to be incompatible with the results of this analysis, as the Monod-Wyman-Changeux parameters derived from the same experimental data demand free energy couplings of an order higher than the second.
Jameson DM, Gratton E, Weber G, Alpert B.
Oxygen distribution and migration within Mbdes Fe and Hbdes Fe: multifrequency phase and modulation fluorometry study.
Biophys J. 1984; 45(4): 795-803. PMCID: PMC1434899Quenching of the intensity and lifetime of porphyrin fluorescence from Mbdes Fe and Hbdes Fe (iron-free myoglobin and hemoglobin) by oxygen was investigated using a multifrequency cross-correlation phase fluorometer. The single exponential decay characteristic of porphyrin emission of Mbdes Fe and Hbdes Fe became doubly exponential upon application of oxygen pressure. The results were interpreted in terms of a general model of dynamic quenching of fluorescence in globular proteins. The model accounted for the rate k+ of acquisition of quencher by the protein, the exit rate k- of quencher from the protein, and the migration rate chi of quencher in the protein interior. The values of k+, k-, and chi were different for Mbdes Fe and Hbdes Fe. The addition of 40% sucrose, which increased the bulk viscosity sixfold, modified these rates. These results are discussed and compared with previous quenching studies on proteins. The significance of these results and ... [truncated at 150 words]
Gratton E, Jameson DM, Weber G, Alpert B.
A model of dynamic quenching of fluorescence in globular proteins.
Biophys J. 1984; 45(4): 789-94. PMCID: PMC1434901A model is presented for the quenching of a fluorophore in a protein interior. At low quencher concentration the quenching process is determined by the acquisition rate of quencher by the protein, the migration rate of quencher in the protein interior, and the exit rate of quencher from the protein. In cases where the fluorescence emission observed in the absence of quencher could be described by a single exponential decay, the presence of quencher led to doubly exponential decay times, and the aforementioned exit rates of the quencher could be determined from experimental data. At high quencher concentration, the processes became more complex, and the deterministic rate equations used at low quencher concentration had to be modified to take into account the Poisson distribution of quencher molecules throughout the protein ensemble and also by using a migration rate for quencher in the protein interior that is a function of the ... [truncated at 150 words]
Gratton E, Jameson DM, Rosato N, Weber G.
Multifrequency cross-correlation phase fluorometer using synchroton radiation.
Rev Sci Instrum. 1984; 55(4): 486-494.The construction and operation of a cross-correlation phase and modulation fluorometer using the synchrotron radiation facility at the ADONE–Frascati electron storage ring is described. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. Use of cross-correlation techniques in conjunction with such a light source permits one to isolate one harmonic frequency from the adjacent frequencies with high precision. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using two coupled frequency synthesizers, one of which drives the radio-frequency cavity of the storage ring and the other which modulates the response of the photomultipliers used for the signal detection. The accuracy, reproducibility, and sensitivity of the instrumentation have been determined on a number of systems and are reported.
Gratton E, Jameson DM, Rosato N, Weber G.
Multifrequency cross-correlation phase fluorometry using synchrotron radiation.
29th Annual Meeting of the Biophysical Society, San Antonio, Texas, February 19-23, 1984.
Biophys J. 1984; 45(2): 321a, W-Pos58.The construction and operation of a cross-correlation phase and modulation fluorometer using the synchrotron radiation facility at the ADONE-Frascati electron storage ring is described. In the frequency domain the pulsed source gives a large series of equally spaced harmonic frequencies. Use of cross-correlation techniques in conjunction with a high repetition rate pulsed light source permits one to isolate one harmonic frequency from the adjacent frequencies with high precision. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using two coupled frequency synthesizers, one of which drives the radiofrequency cavity of the storage ring and the other which modulates the response of the photomultipliers used for the signal detection. The accuracy, reproducibility and sensitivity of the instrumentation have been determined experimentally. A study of measurement artifacts related to the color error of the photomultipliers has been carried out and no sensible color error was ... [truncated at 150 words]
Scarlata SF, Rholam M, Weber G.
Local motions in proteins as investigated by the thermal coefficient of the frictional resistance to rotation.
29th Annual Meeting of the Biophysical Society, San Antonio, Texas, February 19-23, 1984.
Biophys J. 1984; 45(2): 380a, W-Pos224.We have measured the thermal coefficient of the frictional resistance to rotation (a) through the fluorescence polarization of Tryptophan and Tyrosine both free in solution and as intrinsic protein chromophores. For free fluorophores (Tyr, Trp, PRODAN and Perylene) we found: 1) a is a function of the solvent viscosity only and is independent of the chromophore used in its measurement, 2) The values of a are equal to those determined by flow viscometry. In proteins we observe two distinct values of a; one at lower temperatures (αs) equal to the thermal coefficient of the solvent viscosity and at higher temperatures a second, reduced value (αp) is seen, the magnitude of which is distinctive of the individual protein. The transition from αs to αp occurs at the rotational amplitude at which the motions of the chromophore become limited by the surrounding peptide. The magnitude of αp appears to depend on ... [truncated at 150 words]
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Pressure-dissociation and enzyme activity of lactate dehydrogenases: conformational drift of the dissociated monomers.
29th Annual Meeting of the Biophysical Society, San Antonio, Texas, February 19-23, 1984.
Biophys J. 1984; 45(2): 171a, T-PM-E3.We have studied the dissociation under hydrostatic pressure of porcine and bovine lactate dehydrogenase (H4) by methods previously used by Paladini and Weber [Paladini, A. and Weber, G. (1981) Biochemistry 20, 2587-2593]. The results show that hydrostatic pressures, in the range of lbar-3Kbar, promotes the dissociation of these proteins in solution of 10 ig/ml to 1 mg/ml concentration (0.07 to 7 PM tetramer). The standard volume changes upon dissociation were in the range of 220 ml/mole to 280 ml/mole. The reversibility of the pressure effects was better than 95% as judged by recovery of either excitation-polarization spectrum of the intrinsic protein fluorescence or of the dansyl polarization. The initial polarization was regained upon release of the pressure, but recovery of enzyme activity took one hour to several days depending upon the highest pressure applied and the duration of pressure incubation, in agreement with previous work of Jaenicke et. al. [M4ller, ... [truncated at 150 words]
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Pressure dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in single-component phosphatidylcholine liposomes.
Biochemistry. 1983; 22(24): 5544-5550.Small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) of dimyristoyl-L-α-phosphatidylcholine (DMPC) and dipalmitoyl-L-α-phosphatidylcholine (DPPC) and the MLV of dioleoyl-L-α-phosphatidylcholine(DOPC) were examined by steady-state polarization fluorometry under pressure in the range 10-3 - 2 kbar. Isothermal pressure induced phase transitions were observed in DPPC and DMPC vesicles incorporated with 1,6-diphenyl-1,3,5-hexatriene. The temperature to pressure equivalences, dT/dP, estimated from the transition point, are 29.5 °C kbar-' for DPPC(SUV), 22.7 °C/kbar for DPPC(MLV), 22.2 °C/kbar for DMPC(SUV), and 25.9 °C/kbar for DMPC(MLV) in an aqueous phase containing 0.1 M KCl and 0.01 M tris(hydroxymethy1)-aminomethane at pH 8.2. Even though there is no phase transition, we are still able to estimate a dT/dP of about 21 °C/kbar for DOPC(MLV). All the values of dT/dP obtained from this study are within the range typical for lipidinvolving processes. The fluidity changes in the pretransition in DMPC(MLV) and in DPPC(MLV) observed in isobaric temperature studies are not ... [truncated at 150 words]
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Old and new developments in fluorescence spectroscopy.
NATO Advanced Study Institute on Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology. St. Andrews, Scotland. March 16-24, 1980.
Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology (NATO ASI Series A, Vol. 69). By RB Cundall and RE Dale (Editors). Plenum Press, pp. 1-22, 1983. ISBN: 0306414767Introduction: Recent years have seen remarkable advances in the measurement of the properties of fluorescent solutions and of the more complex fluorescent biological systems. These advances have followed progress in optics and electronics including the introduction of instrumentation with digital output that allows a much better evaluation of results, and it is easy to forget how long ago the theoretical bases of the subject were laid down. In fact the fundamental observations on all the aspects of fluorescence capable of yielding information on molecular properties had already been made by 1930. Recent advances have extended our ability to study these properties to virtually every system, while the original observations referred to exceptionally favourable materials and circumstances. I shall briefly refer to what I consider to be the four most important discoveries in this area...
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Stability of oligomeric proteins and its bearing on their association equilibria (a reply).
Proc Natl Acad Sci USA. 1983; 80(17): 5303-5304. PMCID: PMC384243
Younis HM, Weber G, Boyer JS.
Activity and conformational changes in chloroplast coupling factor induced by ion binding: formation of a magnesium-enzyme-phosphate complex.
Biochemistry. 1983; 22(10): 2505-2512.The effects of ions on the conformation and activity of chloroplast coupling factor (CF,) were studied by using tryptophan as an intrinsic fluorescence probe in CF1. The fluorescence of tryptophan decreased when MgCl2 or sodium phosphate was added to the protein. The decrease indicated that Mg2+ and inorganic phosphate (Pi) bound directly to the protein. The decrease saturated at 1.2 mM Mg2+ and at 0.8 mM Pi, although Pi showed evidence of a higher affinity site saturating around 80 μM. The decrease in fluorescence could also be observed when Pi was added after addition of Mg2+, which indicated that a ternary complex of Mg2+-CF1-Pi formed. If the reverse addition sequence was used, a ternary complex was not observed. The free energy of dissociation for each ion added singly was 4.6, 6.2, and 4.8 kcal/mol for Mg2+, Pi (high-affinity site), and Pi (low-affinity site), respectively. The magnitude of these free energies ... [truncated at 150 words]
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The effect of high pressure upon proteins and other biomolecules.
Q Rev Biophys. 1983; 16(1): 89-112.Scope of this Review: We shall not attempt here to enumerate the results or review in a systematic way the significant literature dealing with the use of high pressure in studies of proteins and other molecules of biological interest. Two recent reviews on this subject, one by Morild (1981) and another by Herernans (1982), and a further article by Jaenicke (1981) on enzymes under extreme environmental conditions contain expositions and references that would render redundant such a task. Rather we concentrate here on the examination of the conceptual framework employed in the interpretation of high pressure experiments and in the critical discussion of our knowledge of selected areas of present interest and likely future significance.
Chong PLG, Cossins AR, Weber G.
A differential polarized phase fluorometric study of the effects of high hydrostatic pressure upon the fluidity of cellular membranes.
Biochemistry. 1983; 22(2): 409-415.The effects of high hydrostatic pressure (up to 2 kbar) upon the fluidity and order of the synaptic and myelin membrane fractions of goldfish brain have been studied by using steady-state and differential polarized phase fluorometry. Probe motion provided a measure of membrane order (r infinity) and probe rotational rate (R). Membrane order became progressively greater as pressure was increased up to approximately 2 kbar. This effect was similar over the temperature range 5.6-34.3 degrees C. An increase in pressure of 1 kbar had an effect on membrane order that was equivalent to a 13-19 degrees C reduction in temperature. Membrane order was essentially identical during pressurization and depressurization. At 5.6 degrees C, pressurization caused a large increase in R, and similar, though less dramatic, anomalies occurred at higher temperatures. It is suggested that this is due to the segregation of probe molecules in highly ordered membranes, which leads either ... [truncated at 150 words]
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Asymmetric ligand binding by haemoglobin.
Nature. 1982; 300: 603-607.The asymmetry observed in the titration curve of haemoglobin with oxygen is possible only if the α-α and β-β subunit interactions change by different amounts on oxygenation. This necessary difference justifies the α2β2 structure on functional grounds and its magnitude shows that haemoglobin function results from reversible oxygen binding to the β subunit.
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Fluorescence depolarization and rotational modes of tyrosine in bovine pancreatic trypsin inhibitor.
Biochemistry. 1982; 21(23): 5924-5927.The fluorescence of bovine pancreatic trypsin inhibitor (BPTI) is due to one or more of its four tyrosine residues. Observations of the stationary polarization of the fluorescence over a large range of temperatures and viscosities permit the demonstration of at least three modes of tyrosine rotation, and perhaps an ultrafast fourth one. The slowest mode is one of motion of the whole molecule; the second, a much faster motion limited to an amplitude of 11 degrees, is not changed by quenching of the fluorescence through addition of citrate and is therefore ascribed to the motion of internal tyrosines of BPTI. The third mode of motion is faster still; it has an amplitude similar to that of the second and, being sensitive to citrate quenching, is attributed to the rotation of the external tyrosine residue. A residual depolarization corresponding to a rotational amplitude of 22 degrees is deduced by comparison of ... [truncated at 150 words]
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Dynamics and time-averaged chemical potential of proteins: importance in oligomer association.
Proc Natl Acad Sci USA. 1982; 79(17): 5268-5271. PMCID: PMC346877The chemical potential of a protein is a time-averaged quantity whose value depends upon the fractions of time spent in the different conformations and therefore upon the protein dynamics. If the monomer involved in an association equilibrium undergoes unfolding limited by its lifetime, its chemical potential as well as that of the associated form will not be constant and the free energy of association will be a diminishing function of the degree of dissociation. For a unique free energy of association, the logarithm of the protein concentration must change by 2.86 units to increase the degree of dissociation from 0.1 to 0.9. The dissociation of enolase appears to take place over a significantly smaller range (1.7 units), and dansyl conjugates of enolase show an even narrower range (0.9 unit). A simple descriptive theory is developed, and this shows that the values observed are explained by a difference in free energy ... [truncated at 150 words]
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Properties of S100 protein studied by fluorescence methods.
Biochim Biophys Acta. 1982; 703(2): 231-240.The intrinsic fluorescence of the S100 protein, due to both tyrosine and tryptophan, increases several-fold, in reversible fashion, in solutions at pH 3.0 in comparison with the neutral molecule. The study of the rotational diffusion of the photo-conjugates of l-azidonaphthalene-5-sulfonate with S100 as a function of pH, the concentration-dependence of the fluorescence polarization and the electrophoretic patterns indicate that protein unfolding without dissociation into subunits takes place in the pH region 4-3.4 and that dissociation into subunits is complete in μM solutions of the protein at pH 2.9. Anionic binding sites, probably connected with arginine residues, can be detected in acid solutions, as indicated by a 30-fold increase in fluorescence efficiency of added anilinonaphthalene sulfonate at pH 3.4, as compared to the efficiency at neutral pH. At pH 3.4 the protein can be transferred reversibly to an isobutanol or pentanol phase, not only by the addition of sodium p-toluenesulfonate, which ... [truncated at 150 words]
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Interaction of bovine S100 protein with detergents and phosphatidylcholine vesicles.
Biochim Biophys Acta. 1982; 703(2): 241-246.The interaction of bovine S100 protein with sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), octaethyleneglycol dodecyl ether (OEGDE), and egg phosphatidylcholine vesicles was followed by fluorescence methods. The monomeric forms of both anionic (SDS) and cationic (CTAB) detergents interact with the protein, producing changes in the tryptophan and tyrosine fluorescence similar to those seen when the protein is exposed to low or high pH. No additional interaction was detected between the S100 protein and the micelles for the charged detergents. No interactions were detected with either the monomeric form or the micelles of the non-ionic detergent OEGDE. Fluorescence polarization studies of S100 protein photo-labelled with azidonaphthalene sulfonate show that the protein binds to egg phosphatidylcholine vesicles, even at neutral pH and in the absence of bivalent or monovalent cations and that the strength of the binding of the protein to the vesicles increases at acid pH. At neutralpH binding of ... [truncated at 150 words]
Chong PLG, Jameson DM, Fortes PAG, Weber G.
Effect of pressure and temperature on (Na+ + K+)-ATPase.
28th Annual Meeting of the Biophysical Society, Boston, Massachusetts, February 14-17, 1982.
Biophys J. 1982; 37(2): 148, M-PM-Po92.The inhibition of (Na+ + K+)-ATPase activities by pressure has been reported by Smedt et al. ((1979) Biochem. Biophys. Acta, 556, 479) who suggested that it was due to changes in membrane fluidity. Dog kidney (Na+ + K+)-ATPase was purified by the method of J6rgensen. The enzyme activities were measured under hydrostatic pressures in the range of 10-3 to 2.5 Kbar. The K-pNPPase activity decreases with pressure with an apparent activation volume of 17 ml/mole at 35.50 C. The dT/dP value for this enzyme reaction is about 22° Kbar-1. The Na-K ATPase activity also decreases with pressure with a dT/dP value estimated to be 16 to 200 Kbar-1. The observed dT/dP values suggest that both activities are linked to lipid-involving processes. The enzyme activities were then correlated with membrane fluidity by measurements of the change of stationary fluorescence polarization of diphenylhexatriene (DPH) in ATPase membranes under pressure which itself showed ... [truncated at 150 words]
Chryssomallis GS, Torgerson PM, Drickamer HG, Weber G.
Effect of hydrostatic pressure on lysozyme and chymotrypsinogen detected by fluorescence polarization.
Biochemistry. 1981; 20(14): 3955-3959.The effect of hydrostatic pressure upon solutions of chymotrypsinogen and lysozyme at room temperature has been followed by employing a new technique [Chryssomallis, G. S., Drickamer, H. G., & Weber, G. (1978) J. Appl. Phys. 49, 3084] that permits the measurement of fluorescence polarization at pressures of up to 10 kbar. Lysozyme shows a stable, reversible 60% increase in apparent volume when the pressure is raised to 9 kbar. This can be given a simple interpretation in terms of solvent penetration of the structure at higher pressures. In contrast, the results with chymotrypsinogen are time dependent and only partially reversible on release of the pressure. They involve conversion (tl/e = 5 min) to a form with a lower rotational rate at approximately 6 kbar and return to a fast-rotating form at higher pressure. This latter form persists on pressure release. The possibility of generating what are clearly metastable conformations, not ... [truncated at 150 words]
MacGregor RB Jr, Weber G.
Fluorophores in polar media: spectral effects of the Langevin distribution of electrostatic interactions.
Luminescence from Biological and Synthetic Macromolecules: Eighth Katzir Conference (Annals of the New York Academy of Sciences, Vol. 366). pp. 140-154, 1981. Introduction: The models employed for the analysis of the dependence of the fluorescence spectrum upon the dielectric properties of the consider the latter as a continuum which causes changes in the electronic energy levels through the interaction of the fluorophore with the reaction field that it induces upon its dielectric environment. In models of this kind, the change in energy of the excited state with respect to an unperturbed state is given by an expression of the for: ...
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Pressure-induced reversible dissociation of enolase.
Biochemistry. 1981; 20(9): 2587-25.A study of the polarization of the intrinsic fluorescence and the fluorescence of dansyl conjugates of enolase shows that an increase in hydrostatic pressure, in the range of 1 bar-3 kbar, promotes the dissociation of this protein into dimers. The dissociation of oligomeric proteins under pressure is predicted to be a general phenomenon by a model that assumes the existence of small "free volumes" at the intersubunit boundaries. The same model predicts a dependence of the standard volume change in the dissociation reaction upon the pressure, owing to the additional surface compressibility of the monomers, and numerical analysis of the results clearly shows that dependence for enolase. For a midpoint dissociation pressure of 1.5 kbar the standard volume change in the dissociation reaction is delta V p0 = -65 ± 8 mL mol-1 and the dependence of the volume change upon pressure (dVp0/dp) is approximately -30 mL mol-1 kbar-1. The ... [truncated at 150 words]
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Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements.
J Phys Chem. 1981; 85(8): 949-953.A closed-form procedure is described for the determination of the decay constants and the relative contributing intensities of the N independent components of a heterogeneous fluorescence emission employing measurements of the phase shift and relative modulation of the total fluorescence at N appropriate harmonic excitation frequencies. At each frequency the phase and modulation measurements yield the real part of the Fourier transform of the fluorescence impulse response, G, and its imaginary part, S. It is shown that the moments of a distribution of the lifetimes are linear combinations of the Gs (zero and even moments) or the Ss (odd moments), and the rule for the construction of the coefficients of G and S in these linear combinations is derived. The classical de Prony method is used to obtain the lifetimes and fractional contributions of the components from the moments. For binary and ternary mixtures the numerical computations required are ... [truncated at 150 words]
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Resolution of the pH-dependent heterogeneous fluorescence decay of tryptophan by phase and modulation measurements.
J Phys Chem. 1981; 85(8): 953-958.The tryptophan fluorescence emission arises from the zwitterion and the anion, present in amounts determined by the pH of the solution. These forms interconvert in times much longer than the fluorescence lifetime, and their absorption and emission spectra are similar enough to make this an ideal binary system to test the resolution procedure by means of phase and modulation measurements at two excitation frequencies. Measurements were made by employing the excitation frequencies of 6,18, and 30 mHz, in the pH range 8-10, in which the relative zwitterion contribution varies from 0.82 to 0.09. Best resolution was expected and achieved by combining the data at 6 and 30 mHz. Resolved lifetimes were within ±0.4 ns of the true lifetimes (3.1, zwitterion; 8.7, anion), and fractional contributions were within 10-20% of expectancy. Such dispersion is predicted for phase and modulation measured lifetimes with standard deviations of ~±50 ps, which in ... [truncated at 150 words]
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Absolute measurements of fluorescence polarization at high pressures.
Rev Sci Instrum. 1981; 52(3): 419-427.The measurement of polarization of the fluorescence of solutions under high pressure is rendered uncertain by the photoelastic birefringency of the windows, and needs a correction dependent upon the fraction of the light emerging from a window with polarization normal to that of the incident light (scrambling coefficient). This correction, which increases rapidly with applied pressure, differs according to whether the method of measurement employs rotation of the excitation polarizer (T format) or the emission polarizer (L format), and the latter is shown to involve the more reliable and the smaller correction. A four window pressure bomb housing a 1 ml volume cuvette that permits absorption and fluorescence measurements up to 4 kbar is described. The polarization correction up to 2.5 kbar was determined. Its reliability was demonstrated by measurements of the fluorescence polarization spectra of fluorophores in solvents of high viscosity at all pressures and by comparison of isopiestic ... [truncated at 150 words]
Torgerson PM, Drickamer HG, Weber G.
Effect of hydrostatic pressure upon ethidium bromide association with transfer ribonucleic acid.
Biochemistry. 1980; 19(17): 3957-3960.The binding of ethidium bromide to yeast phenylalanine-specific transfer ribonucleic acid (tRNAPhe) has been investigated in the pressure range from 1 atm to 9 kbar in the presence of 100 mM sodium chloride and 10 mM magnesium chloride, pH 7.7. One high-affinity binding site for ethidium is present, with a dissociation constant of 2.4 × 10(-6) M at 1 atm and 22 degrees C. Binding to this site is enhanced with increasing pressure, the dissociation constant reaching 2.9 × 10(-7) M at 2 kbar. Pressure also promotes the binding of ethidium to lower affinity sites of tRNAPhe. The standard volume change upon complex formation is found to be 25.6 ± mL/mol for the first ethidium bound. If sodium is replaced by lithium in the buffer, the standard volume change is 23.3 ± 0.5 mL/mol. We conclude that decrease of the electrostatic repulsion in the negatively charged tRNAPhe by binding of ... [truncated at 150 words]
Grigorova AM, Cittanova N, Weber G.
Existence of multiple sites for ANS in an alpha-fetoprotein fraction demonstration by fluorescence polarization.
Biochem Biophys Res Comm. 1980; 94(2): 413-418.Voss EW Jr, Watt RM, Weber G.
Solvent perturbation of the fluorescence of fluorescyl ligand bound to specific antibody. Fluorescence enhancement of antibody bound fluorescein (hapten) in deuterium oxide.
Mol Immunol. 1980; 17(4): 505-517.The relative fluorescence quantum yield (Φ) and fluorescence lifetime (τ) of fluorescein bound to
specifically purified high affinity rabbit and chicken IgG antibody molecules was significantly increased in deuterium oxide (D2O) with respect to the complex in aqueous solution. The degree of enhancement appeared to be dependent on the affinity of the antibody for the ligand, the highest affinity sites showing the smallest increases in quantum yield. Fluorescence enhancement in D2O was identical for ligand bound to IgG and the F(ab)'2 fragments derived from the same molecule. The isotope effect was apparently not due to ligand dissociation, as shown by a comparison of difference spectroscopy and equilibrium dialysis results in D2O and H2O buffers. and by the observation of similar enhancement with affinity labeled antibody. Fluorescence enhancement of antibody bound ligand in D2O was temperature dependent. The relative ΦD2O/ΦH2O of free fluorescein increased, while that of bound ligand remained relatively constant ... [truncated at 150 words]
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Ligand-induced transfer of proteins between phases: dependence upon the strength of ion pair interactions.
Biochemistry. 1980; 19(5): 990-995.Chemical modifications of ionizable groups of bovine serum albumin and lysozyme are described which lessen both pH and solvent polarity restrictions to the transfer of the proteins from water to alcohol phases on addition of suitable anions. Esterification of one-third of the carboxyl groups of albumin with triethyloxonium fluoroborate yielded protein fractions which could be transferred, by addition of toluenesulfonate or octylsulfonate, into butanol at pH 4.8 but not pH 7. I , while the intact protein could only be transferred at pH 2.4. Further, guanidation of 30% of the t-amino groups yielded an ethylated-guanidated albumin which could be transferred into 1-butanol at pH 7.1 or into 1-octanol at pH 2.4. This notable increase in the ease of partition upon guanidation is directly traceable to the higher stability of the neutral ion pairs formed by guanidine (pK ~ 13) above those formed by the t-amino groups of lysine (pK ~ ... [truncated at 150 words]
Alpert B, Jameson DM, Weber G.
Tryptophan emission from human hemoglobin and its isolated subunits.
Photochem Photobiol. 1980; 31(1): 1-4.The emission spectra of human adult hemoglobin A, and its isolated alpha and beta subunits were obtained using a highly sensitive photon-counting spectrofluorometer. The quantum yields of the emissions, relative to free tryptophan, were also measured as well as the excitation polarization spectra for hemoglobin A. and apohemoglobin. The fluorophore bis-ANS was utilized to probe for the presence of apoproteins in the hemoprotein preparations. The work suggests that tryptophan may be useful as an intrinsic probe to study dynamical processes in hemoglobin.
Lakowicz JR, Freshwater G, Weber G.
Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies.
Biophys J. 1980; 32(1): 591-601. PMCID: PMC1327357Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, alpha-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29 degrees ±6 degrees during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.
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The effects of high-pressure upon proteins and other biomolecules.
Symposium held in honor of Professor Kunio Yagi on the occasion of the 60th birthday anniversary under the auspices of the Japanese Biochemical Society.
New Horizons in Biological Chemistry. By K Yagi, M Koike, N Seikagakkai, and N Seikagakkai (Editors). Japan Scientific Societies Press, pp. 237-245, 1980.
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A new red-edge effect in aromatic molecules: anomaly of apparent rotation revealed by fluorescence polarization.
J Chem Phys. 1978; 69(6): 2393-2400.Measurements of stationary fluorescence polarization at wavelengths of excitation that yield limiting polarizations close to 1/2 and 1/7, respectively, permit the characterization of the rates of in–plane and out-of-plane rotation in aromatic compounds. Propylene glycol solutions of 1- and 2-naphthylamine, some of their derivatives, anthracene, and indole show the expected dependence of the rate of rotation upon limiting polarization, but all of them additionally display on excitation at the red edge of the absorption, a decrease in the rotational rate, which then approaches the rate observed for pure out-of-plane rotations. Differential phase measurements of the polarized components of the fluorescence of 1-naphthylamine confirm this effect. After careful check of the effects of temperature and excitation wavelength upon the emission spectrum and the fluorescence lifetimes, it is concluded that the red-edge rotational anomaly arises from the existence of out-of-plane transition moments in absorption and emission in this spectral region. The physical ... [truncated at 150 words]
Farris FJ, Weber G, Chiang CC, Paul IC.
Preparation, crystalline structure, and spectral properties of the fluorescent probe 4,4'-bis-1-phenylamino-8-naphthalenesulfonate.
J Am Chem Soc. 1978; 100(14): 4469-4474.A method for the preparation and purification of the covalent dimers of 1 -phenylamino- and I -toluidylaminonaphthalenesulfonic acids is described. X-ray analysis of the crystals of the potassium salt of the former (bis-ANS) showed this to be 4,4'-bis- 1 -phenylaminonaphthalene-8-sulfonateB. y similarity of the NMR spectra and optical properties the second dimer is recognized to be 4,4'-bis- 1 -toluidylaminonaphthalene-8-sulfoniacc id. NMR spectra, molar absorption coefficients, fluorescent lifetimes, and the detailed structural parameters derived from the x-ray data are presented. Some of the uses of the compounds as fluorescent probes in solutions and crystals of proteins are briefly discussed.
Chryssomallis GS, Drickamer HG, Weber G.
The measurement of fluorescence polarization at high pressure.
J Appl Phys. 1978; 49(6): 3084-3987.High pressure was used to change the viscosity isothermally for the measurement of polarization of fluorescence of small fluorescent molecules in solution. A new technique, necessary for the measurements, as well as a new instrument to perform them, are described and discussed. Although the apparatus is not well suited for determining absolute polarization values, it is quite accurate in detecting changes in polarization due to change in pressure. Independent readings of polarization at 1 atm were therefore obtained using a different instrument. For the sample system, quinine sulfate in isobutanol, there is good agreement between the data obtained by pressure variation and those obtained by temperature variation. The extension of the method to study polymers and proteins in solution and its advantages over previous methods are also discussed.
Jameson DM, Weber G, Spencer RD, Mitchell GW.
Fluorescence polarization: measurements with a photon-counting photometer.
Rev Sci Instrum. 1978; 49(4): 510-514.A T-format, photon-counting polarization photometer was constructed to facilitate precise polarization measurements at low signal-to-noise ratios. The instrument optics and electronics are described. Several examples of the instrument's performance are given, including measurements on picomolar fluorescein solutions at low excitation resolution and the excitation polarization spectrum of indole (2×10–4M) at a resolution of 5 Å.
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Ligand-promoted transfer of proteins between phases: spontaneous and electrically helped.
Proc Natl Acad Sci USA. 1978; 75(2): 779-783. PMCID: PMC411340A model system for the partitioning of peripheral membrane proteins into membranes by ligand binding has been examined experimentally. Both bovine serum albumin and lysozyme partition between water and 1-butanol by the addition of sodium p-toluene sulfonate at pH 2.4. The partitioning is characterized by high orders of reaction: 25 and 10, respectively. Theory indicates that these high orders of reaction need not result from cooperative ligand binding in either phase, but depend primarily upon the number N of protein sites at which the transfer-promoting ligand binds, and on the difference in free energy of formation δF0s of the protein-ligand complexes in the two phases. From the reaction orders and the experimental values of N, 80 for albumin and 11 for lysozyme, δF0s was calculated to be -0.5 kcal/mol (-2.1 kJ/mol) and -0.8 kcal/mol (-2.5 kJ/mol) per ligand bound, respectively. Experiments measuring the dependence on ligand concentration of the rate ... [truncated at 150 words]
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Limited rotational motion: recognition by differential phase fluorometry.
Acta Phys Pol. 1978; A54: 859-865.The rotational motions of fluorophores giving rise to a limited decay of the fluorescence polarization can be detected by measurements of diffrrential phase fluorometry. Like anisotropic rotations or, in general, rotations owing to more than one rotational rate, limited rotations give rise to a deficit in the maximum differential delay of the polarized components (tangent defect). It is shown that the amplitude allowed to the rotations, and the absolute rotational rate of a fluorophore of known lifetime and limiting polarization placed in an unknown medium can be extracted from the experimental data.
Visser AJWG, Li TM, Drickamer HG, Weber G.
Volume changes in the formation of internal complexes of flavinyltryptophan peptides.
Biochemistry. 1977; 16(22): 4883-4886.The effect of pressure, up to 10 kbar, on the fluorescence yield and lifetime of two flavinyltryptophan peptides was investigated. These peptides differed only in the number of methylene groups, respectively three and five, separating the chromophores. At atmospheric pressure the closed nonfluorescent form predominated in both compounds constitutin 94% of the total in the short-linked peptide and 80% in the long-linked one. The fluorescence of both peptides decreased at high pressure and the volume change upon formation of the nonfluorescent complex in the short peptide (-1.8 mL/mol) was less than half of the change in the long peptide (-4.8 mL/mol) or the value for FAD (-4.3 mL/mol). The much smaller compressibility of the short peptides is attributed to the mechanical constraint to the approach of the interacting rings, imposed by the short link. Mechanical constraints of similar nature may be expected to be operative in proteins. Their importance in ... [truncated at 150 words]
Visser AJWG, Li TM, Drickamer HG, Weber G.
Effect of pressure upon the fluorescence of various flavodoxins.
Biochemistry. 1977; 16(22): 4879-4882.The effects of hydrostatic pressure in the range of 10(-3) to 11 kbar on the fluorescence of flavodoxins from Peptostreptococcus elsdenii, Desulfovibrio vulgaris, Azotobacter vinelandii, and Clostridium MP were investigated. The first three flavoproteins showed under high pressure enhancements of flavin fluorescence of over 50 times resulting from the release of flavin mononucleotide from the protein complex. The Clostridial flavodoxin showed a very much smaller fluorescence change. At pH 7.5 the high-pressure fluorescence changes of the flavodoxins of D. vulgaris and P. elsdenii were not reversed by decompression, but in A. Vinelandii the pressure changes were over 80% reversible. At pH 5 over 80% reversibility was restored to the flavodoxins of D. vulgaris and P. elsdenii, although the pressure dependence of the fluorescence changes was very similar in the reversible and irreversible cases. The midpoint pressures in the reversible reactions were 4.7 kbar (D. vulgaris), 8.7 kbar (P. elsdenii), and ... [truncated at 150 words]
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Rotational anisotropy and solvent-fluorophore bonds: an investigation by differential polarized phase fluorometry.
J Chem Phys. 1977; 66(9): 4092-4099.Differential polarized phase fluorometry has been employed in a comparative study of unsubstituted aromatic hydrocarbons, anthracene, perylene, chrysene, and molecules with similar aromatic skeleton but with several groups that can form hydrogen bonds with the solvent. Strongly anisotropic rotations are demonstrated in the former cases by values of the maximum differential tangent 15–25% smaller than the values characteristic of spherical rotations. On the other hand, molecules having two or more groups that form hydrogen bonds with the solvent show values of the differential tangent that agree with the isotropic rotation value within 1%–2%. In these latter cases the apparent molar volumes computed by Einstein's equation are close to those expected from the molecular mass, but in the unsubstituted aromatics they are anomalously small, in some cases only one-fifth of the volume expected. By comparison with the rotational models discussed in the previous paper it is concluded that the anisotropic rotations ... [truncated at 150 words]
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Resolution of the fluorescence excitation spectrum of indole into the 1La and 1Lb excitations bands.
Photochem Photobiol. 1977; 25: 441-444.The fluorescence excitation spectrum and the excitation polarization spectrum of indole in propylene glycol were measured at -58°C, after selecting by optical filters the emission originating from the 1La electronic level. From the analysis of these spectra, the excitation spectrum was resolved into the 1La, and 1Lb excitation bands. A similar resolution of the excitation spectrum of tryptophan is given. This method can also be applied to the resolution of the emission spectrum in cases of dual emission.
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Theory of differential phase fluorometry: detection of anisotropic molecular rotations.
J Chem Phys. 1977; 66(9): 4081-4091.A general description of transformations in the excited state is employed to derive the general equations for the differential delay and the modulation ratio of the fluorescence owing to different pairs of excited species present. These equations yield directly the differential delay and the modulation ratio of the polarized components of the fluorescence from a rotating spherical molecule. Similar equations for a rotating irregular molecule are then derived from the sine and cosine transforms of the impulse response of the polarized components of the fluorescence emission. It is shown that for excitation at a wavelength at which the limiting polarization is high, namely, 1/2 to 3/11, the maximum differential tangent observed for anisotropic rotations is uniformly lower than the value for the sphere. The magnitude of this tangent defect permits an estimate of the minimum standard deviation of the principal rotational rates of the ellipsoid of inertia that describes the ... [truncated at 150 words]
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Anisotropic rotations in 1-naphthylamine. Existence of a red-edge transition moment normal to the ring plane.
Chem Phys Lett. 1977; 45(1): 140-144.The rotational motions of 1-naphthylamine in propylene glycol are investigated by means of steady-state flourescence polarization measurements and differential polarized phase flourometry, on excitation at various wavelengths. For excitation at 370 nm the average rotational rate is faster than for excitation at shorter wavelength and the rotations are clearly anisotropic. On excitation from 370nm to the red edge of the spectrum (390 nm) the average rotational rate slows down by a factor of two and the rotations become nearly isotropic. The results reveal the possible existence of an excited state generated preferentially by excitation at the edge of the absorption, in which the transition moments in both absorption and emission are prependicular to the plane of the aromatic rings.
Li TM, Hook JW, Drickamer HG, Weber G.
Plurality of pressure-denatured forms in lysozyme and chymotrypsinogen.
Biochemistry. 1976; 15(25): 5571-5580.The ultraviolet fluorescence of lysozyme shows a two-step decrease in efficiency when the pressure of the solution is raised from 10-3 to 11 kbars. The fluorescence of chymotrypsinogen decreases smoothly in a single step confined to the region below 8 kbars, with a midpoint at 5.8 kbars. At 11 kbars, lysozyme binds 1 mol of 8-anilino-1-naphthalenesulfonate (ANS) with a dissociation constant = 4.8 μM. Chymotrypsinogen binds 2 mol with a dissociation constant = 14 μM and Hill coefficient close to unity. In both proteins, binding of ANS is confined to the region above 6 kbars. In
lysozyme it roughly corresponds to the second step of ultraviolet fluorescence changes. In chymotrypsinogen it has only minimal overlap with the region of protein-fluorescence change. The existence of two distinct regions of change of ultraviolet fluorescence in lysozyme and the disparity of the regions of change of protein fluorescence and of ANS binding ... [truncated at 150 words]
Weber G, Helgerson SL, Cramer WA, Mitchell GW.
Changes in rotational motion of a cell-bound fluorophore caused by colicin E1: a study by fluorescence polarization and differential polarized phase fluorometry.
Biochemistry. 1976; 15(20): 4429-4432.The stationary fluorescence polarization and the differential phase delay of the polarized components of the fluorescence of 1-phenylnaphthylamine in Escherichia coli suspensions were measured before and after addition of colicin E1. Both sets of measurements register an increase in the rotational relaxation time of the fluorescent probe when colicin is present. These increases are absent in an E. coli mutant tolerant to colicin E1. The physical interpretation of the changes demands separate estimation of the fraction f2 of the emitting fluorophores that change their properties upon colicin addition and of the rotational relaxation time p2 of this fraction, following the colicin-induced changes. By themselves, the steady state polarizaiton observations permit only the conclusion that f2 must be in the range of 1-0.06 and the change in p2/pi between 1.5 and a value larger than 10. Combination of the data of stationary polarization with those of differential phase fluorometry results in ... [truncated at 150 words]
Jameson DM, Spencer RD, Weber G.
Construction and performance of a scanning, photon-counting spectrofluorometer.
Rev Sci Instrum. 1976; 47(9): 1038-8.A photon-counting spectrofluorometer interfaced to a Nuclear Data ND 812 computer was constructed to facilitate acquisition of emission spectra at very low signal-to-noise ratios. The photon-counting technique combined with repetitive scanning permits the detection and quantitation of emission from strong fluorophors near the picomolar level. The computer performs a variety of digital manipulations such as subtraction of background, normalization of spectra, corrections for instrument response, and polarization calculations. Special attention needs to be given to handling of cuvets and solvents to minimize background signals.
Li TM, Hook JW, Drickamer HG, Weber G.
Effects of pressure upon the fluorescence of the riboflavin binding protein and its flavin mononucleotide complex.
Biochemistry. 1976; 15(15): 3205-3211.The effect of pressure in the range of 10(-3)-10 kbars upon the ultraviolet fluorescence of the riboflavin binding protein and the fluorescences of its complex with flavin mononucleotide has been studied. The fluorescence spectrum of the isolated protein showed a reversible red shift of 12nm (1000 cm-1) at high pressure, indicating the reversible exposure of the tryptophan to solvent. From the pressure dependence of the visible fluorescence of the protein-flavin complex in the region of 1-4 kbars the volume change in dissociation of the protein-ligand complex was estimated to be +3.3ml/mol. A very sharp increase in fluorescence-up to 30-fold of the low-pressure value-takes place in the region 5-8 kbars. This increase is due to release of the flavin from the complex and is assigned to pressure denaturation of the protein. The midpoint, rho 1/2, of this transition was found at 6.5 kbars and the change in volume, delta, in the ... [truncated at 150 words]
Secrist JA, Barrio JR, Leonard NJ, Weber G.
Fluorescent derivatives of adenine-containing compounds.
Patent US3960840, 01 Jun 1976.Fluorescent analogs of biologically active coenzymes are made by reaction of certain adenine-containing coenzymes with acetaldehyde. The reaction products retain a substantial portion of their biologic activity and are fluorescent in the visible range under ultraviolet illumination.
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Demonstration of anisotropic molecular rotations by differential polarized phase fluorometry.
International Conference on Excited States of Biological Molecules at the Calouste Gulbenkian Foundation Centre, Lisbon, Portugal, on April 18-24, 1974.
Excited States of Biological Molecules (Wiley Monographs in Chemical Physics). By JB Birks (Editors). John Wiley & Sons, pp. 72-76, 1976. ISBN: 9780471074137
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What we have learnt about proteins from the study of their photoexcited states.
International Conference on Excited States of Biological Molecules at the Calouste Gulbenkian Foundation Centre, Lisbon, Portugal, on April 18-24, 1974.
Excited States of Biological Molecules (Wiley Monographs in Chemical Physics). By JB Birks (Editors). John Wiley & Sons, pp. 363-374, 1976. ISBN: 9780471074137
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Practical applications and philosophy of optical spectroscopic probes.
Horizons in Biochemistry and Biophysics (Vol. 2). By E Quagliariello, F Palmieri, and TP Singer (Editors). Addison Wesley, pp. 163-198, 1976.
Schuldiner S, Spencer RD, Weber G, Weil R, Kaback HR.
Lifetime and rotational relaxation time of dansylgalactoside bound to the Lac carrier protein.
J Biol Chem. 1975; 250(23): 8893-8896.The results presented in this paper demonstrate that the excited state lifetime, anisotropy, and rotational relaxation time of 2'-(N-dansyl)aninoethyl 1-thio-beta-D-galactopyranoside (DG2) increase when the probe is bound specifically to the lac carrier protein in "energized" Escherichia coli membrane vesicles. Although the probe also binds nonspecifically to the vesicle membrane, such binding is independent of the lac carrier protein and is unaffected by "energization" of the vesicles. The experiments provide further evidence that the dansylgalactosides are useful probes for the beta-galactoside transport system and support the hypothesis that the changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect changes in the binding of the probe.
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Cooperativity of binding of anilinonaphthalenesulfonate to serum albumin induced by a second ligand.
Biochemistry. 1975; 14(20): 4476-4481.When a ligand X is multiply bound to energetically identical, noninteracting sites of a protein, cooperative binding of this ligand can be induced by the presence of a second ligand Y. This effect should appear whenever multiple interactions exist between the bound X and Y ligands, and vanish when the concentration of Y is made sufficiently large to ensure Y saturation at all concentrations of X. These predictions have been verified for the binding of 8-anilino-1-naphthalenesulfonate to serum albumin, when Y, the effector ion, is 3,5-dihydroxybenzoate. In the presence of 2mM dihydroxybenzoate, the Hill coefficient for anilinonaphthalenesulfonate binding rose steadily from 1 to 1.5 as the number of molecules of ligand bound increased from 1 to 3.3 per albumin molecule. The theory of interactions between isolated ligands, applied in the previous paper (D. A. Kolb and G. Weber (1975), Biochemistry, preceding paper in this issue), is extended to cases of ... [truncated at 150 words]
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Quantitative demonstration of the reciprocity of ligand effects in the ternary complex of chicken heart lactate dehydrogenase with nicotinamide adenine dinucleotide and oxalate.
Biochemistry. 1975; 14(20): 4471-4475.The reciprocity of effects of two ligands simultaneously bound to a protein as a ternary complex may be proven by measurements of four standard free energies of binding. Two of these are for the binding of each ligand in the absence of the other, and the other two for the binding of each ligand in the presence of saturating amounts of the other (conditional free energies). These four quantities have been measured for the complexes of oxalate and nicotinamide adenine dinucleotide with chick heart lactate dehydrogenase. The differences between conditional and unconditional free energies are: oxalate, -1.1 ± 0.3 kcal; NADH,-1.3 ± 0.2 kcal, thus proving the reciprocity within experimental error.
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Energetics of ligand binding to proteins.
Mechanisms and Pathways of Heterotrimeric G Protein Signaling (Advances in Protein Chemistry, Vol. 29). By CB Anfinsen, JT Edsall, and FM Richards (Editors). pp. 1-83, 1975. ISBN: 9780120342297The interaction of proteins with small ligands embodies the fundamental physicochemical principles that are operative in the physiological regulation of enzyme activity, the specific interactions among proteins and of proteins with nucleic acids and other macromolecules. This chapter describes the multiple ligand binding by proteins, binding by multimer proteins, extension of the concept of ligand interaction to covalent bond exchange, cooperativity and ligand correlation, and biological specificity and ligand binding. Many parts of the compact protein structure can contribute toward the shift in energy and structure taking place on ligand binding. The energies of ligand–ligand interaction, although small are sufficient to shift the covalent equilibria in which proteins take part, to a significant extent, and this may be found responsible for the interconversion of chemical and osmotic energies in metabolism. While, the ligand correlation because of the existence of free-energy coupling among the bound ligands is a molecular property to ... [truncated at 150 words]
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Ligand interactions in globular proteins.
A symposium held at the University of Konstanz, West Germany, Sept. 2-6, 1974.
Protein-Ligand Interactions. By H Sund and G Blauer (Editors). Walter de Gruyter, pp. 15-26, 1975. ISBN: 9783110048810
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Optical spectroscopy of biomolecules.
Biochemistry 494. University of Illinois at Urbana-Champaign. Spring semester 1975.
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Polarized fluorescence.
Quantitative Fluorescence Techniques as Applied to Cell Biology. Battelle Research Center, Seattle, Wash., March 27-31, 1972.
Fluorescence Techniques in Cell Biology. By AA Thaer and M Sernetz (Editors). Springer-Verlag New York, LLC, pp. 5-13, 1973. ISBN: 9780387064215
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Synthesis and characterization of two fluorescent sulfhydryl reagents.
Biochemistry. 1973; 12(21): 4154-4161.Two fluorescent sulfhydryl reagents, N-(iodoacetylaminoethyl)-5-naphthylamine-l-sulfonic acid (1,5-IAEDANS) and the 1,8 isomer (l&I-AEDANS), were synthesized. Although sensitive to photocatalyzed degradation, these reagents readily react with thiol compounds and sulfhydryl groups in proteins yielding photostable covalent derivatives. The synthesis, fluorescence spectra, quantum yields, and lifetimes of model compounds in various solvents are presented in this paper. A following paper will report some of the general properties of the protein conjugates.
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Quenching of fluorescence by oxygen. Probe for structural fluctuations in macromolecules.
Biochemistry. 1973; 12(21): 4161-4170.Quenching of the fluorescence of various fluorophores by molecular oxygen has been studied in aqueous and nonaqueous solutions equilibrated with oxygen pressures up to 100 atm. Temperature dependence of quenching, agreement with the Stern-Volmer equation, and fluorescence lifetime measurements indicate that essentially all the observed quenching is dynamic and close to the diffusion-controlled limits. Studies of charged polyamino acids containing tryptophan show that oxygen quenching, in contrast to I-, is completely insensitive to charge effects. Ethidium bromide, when intercalated into double helical DNA, is quenched with 1/30th of the efficiency of the free dye in solution. Three dyes bound to bovine serum albumin were also found to be relatively protected from the free diffusion of oxygen. Quenching of intrinsic or bound fluorophores by molecular oxygen is therefore an appropriate method to determine the accessibility to oxygen of regions of the macromolecule surrounding the fluorophore and indirectly the structural fluctuations in ... [truncated at 150 words]
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Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale.
Biochemistry. 1973; 12(21): 4171-4179.Quenching of the tryptophan fluorescence of native proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pressure of 1500 psi. Measurement of absorption spectra and enzymic activities of protein solutions under these conditions reveal no significant perturbation of the protein structure. The oxygen quenching constant (k+*) for a variety of proteins indicates that the apparent oxygen diffusion rate through the protein matrix is 20-50% of its diffusion rate in water. No tryptophan residues appear to be excluded from quenching, and no correlation of the fluorescence emission maxima with k+* was found, indicating that the rapid oxygen diffusion is present in all regions of the protein, even those normally considered inaccessible to solvent. Energy transfer among tryptophans was excluded as a possible mechanism for the rapid quenching by studies using 305-nm excitation, where energy transfer is known to fail, The dynamic character of ... [truncated at 150 words]
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Subnanosecond solvent relaxation studies by oxygen quenching of fluorescence.
Chem Phys Lett. 1973; 22(2): 419-423.Quenching by oxygen under a pressure of 100 atmospheres reduces the fluorescence lifetime of ethanolic solutions of 1-anilinonaphthalene-8-sulphonate and indole to 54 and 36 psec from the unquenched values of 13.4 and 5.0 nsec, respectively, while the corresponding fluorescence emissions are blue-shifted by 5 and 8 nm. The latter shift, due to incomplete solvent relaxationduring the shortened fluorescence lifetime, is too fast for detection by the complementary technique of nanosecond time-resolved spectroscopy, but is revealed by the quenching method, which provides for a hundred-fold improvement in time-resolution and is capable of wider application.
Barrio JR, Tolman GL, Leonard NJ, Spencer RD, Weber G.
Flavin 1, N 6 -ethenoadenine dinucleotide: dynamic and static quenching of fluorescence.
Proc Natl Acad Sci USA. 1973; 70(3): 941-943. PMCID: PMC433393Flavin 1,N6-ethenoadenine dinucleotide (εFAD) was prepared by the action of chloroacetaldehyde on flavin adenine dinucleotide. This compound, which has two potential fluorophores, ε-adenine and isoalloxazine, shows extremely efficient energy transfer from the former to the latter. The fluorescences of both moieties are greatly diminished in the intact molecule. Determination of the fluorescence yields and lifetimes leads to the conclusion that at 20° in neutral aqueous solution εFAD exists mainly (90%) as an internally complexed or stacked form.
Cogan U, Shinitzky M, Weber G, Nishida T.
Microviscosity and order in the hydrocarbon region of phospholipid and phospholipid-cholesterol dispersions determined with fluorescent probes.
Biochemistry. 1973; 12(3): 521-528.Microviscosity, existence of phase transitions, and structural organization in the hydrocarbon region of lipid dispersions of biological importance were investigated by means of fluorescence polarization techniques. Dispersions of egg lecithin, dipalmitoyllecithin, and lecithin-cholesterol mixtures as well as egg lysolecithin micelles were labeled with perylene, 2-methylanthracene, or 9-vinylanthracene and the microviscosities which represent the harmonic mean of the internal viscosities opposing the in-plane and out-of-plane rotations of the nonpolar fluorescence dyes were evaluated by measuring the degree of the fluorescence depolarization. In the phospholipid systems studied, microviscosity values obtained were lowest for egg lysolecithin, followed by egg lecithin, and were highest for dipalmitoyllecithin. Cholesterol, when incorporated into lecithin dispersions, caused a marked increase in the internal viscosity. A phase transition between liquid crystalline and gel states was detected by determining the dependence of the microviscosity (η) on the absolute temperature (T). Deviation of In η vs. 1/T plots from linearity or ... [truncated at 150 words]
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Kinetic and binding effects of 1, N6-ethenoadenosine triphosphate to aspartate transcarbamylase.
Biochem Biophys Res Comm. 1973; 50(2): 538-543.A fluorescence polarization study reveals that 1,N6-ethenoadenosine triphosphate (εATP) binds to native aspartate transcarbamylase (ATCase) from E. coli. However, unlike adenosine triphosphate (ATP) εATP inhibits the enzyme, which strongly suggests that the N-1 atom in the purine ring is crucial for ATP activation. Study of the binding curve for εATP shows multiple binding sites with overlapping affinities, but a simple system of six equivalent binding sites with a dissociation constant K = 7.5 × 10-5 gives a reasonable approximation to the experimental data.
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Thermodynamics and kinetics of the intramolecular complex in flavin-adenine dinucleotide.
Wenner-Gren Symposium. 23-27 August, 1970. Stockholm, Sweden.
Structure and function of oxidation-reduction enzymes (Vol. 18). By A Åkeson and A Ehrenberg (Editors). Pergamin Press, Oxford-New York, pp. 393-399, 1972. ISBN: 9780080168746INTRODUCTION: The existence of an intramolecular complex in FAD between adenine and isoalloxazine rings (Fig. 1) is now well proven. Fluorescence yield and polarization measurements, absorption spectra, optical rotatory dispersion and circular dichroism spectra, and nuclear magnetic resonance studies can only be interpreted on the assumption of short-range interactions between the purine and alloxazine moieties of the molecule.
The above methods, however, are only capable of assessing characteristics of the time average conformation of the molecules in solution and cannot, without further assumptions, determine the fraction of the total FAD molecules present in a proposed "complex" as opposed to an extended form.
Secrist JA, Barrio JR, Leonard NJ, Weber G.
Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties.
Biochemistry. 1972; 11(19): 3499.The synthesis of fluorescent derivatives of adenosine and cytidine, by reaction with chloroacetaldehyde in aqueous solution at mild pH and temperature, yielding 1,N6-ethenoadenosine hydrochloride and 3,N4-ethenocytidine hydrochloride, respectively, is here described. Analogous derivatives of 3'-AMP, 5'-AMP, 3',5'-cyclic AMP, ADP, ATP, and NAD+ were also synthesized. Observation of the spectroscopic properties of the highly fluorescent adenosine derivatives included the emission maximum for 1,N6-ethenoadenosine at ca. 415 nm (corrected) in buffered aqueous solution at pH 7.0, a quantum yield of 0.56, and a fluorescence lifetime of 20 nsec. Variations in fluorescence with respect to changes in the polarity and viscosity of the solvent and with respect to temperature were also examined, as well as fluorescence excitation and fluorescence polarization spectra. All adenine derivatives had similar fluorescence properties. The 1,N6-etheno-ATP analog showed considerable substrate activity as a replacement of ATP with adenylate kinase, hexokinase, and phosphofructokinase, and exhibited allosteric inhibition of phosphofructokinase. The ... [truncated at 150 words]
Belford GC, Belford RL, Weber G.
Dynamics of fluorescence polarization in macromolecules.
Proc Natl Acad Sci USA. 1972; 69(6): 1392-1393. PMCID: PMC426709Reexamination of the theory of fluorescence time dependence owing to rotational diffusion of rigid macromolecules reveals deficiencies or hidden restrictions in each of the previous treatments. The correct master equation has five exponential decay terms, with preexponential factors that depend upon -the diffusion constants and, in a completely symmetrical fashion, upon the orientations of absorbing and emitting dipoles.
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Ligand binding and internal equilibria in proteins.
Biochemistry. 1972; 11(5): 864-878.An examination is carried out of the relations between the binding of small ligands to proteins (microassociations), the tautomerizations of the peptide chain (conformational changes) and the second- and higher order interactions among the chains (macroassociations). This task is facilitated by the introduction of "apparent" and "conditional" standard free energies. By consideration of the simultaneous binding of two ligands which stabilize different conformations of the peptide chain it is demonstrated that a protein cannot adopt an absolute conformation dependent upon the binding of one ligand in particular. It is also shown that multiple binding of one ligand in the presence of a second ligand can give rise to cooperative effects whether the two types of ligands oppose or enhance each other’s binding. The coupling between the free energies of micro- and macroassociation is introduced by considering a dimer made up of two identical protomers. Coupling between the two association processes ... [truncated at 150 words]
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The reversible association of lysozyme and thyroglobulin. Cooperative binding by near-neighbor interactions.
J Biol Chem. 1972; 247(3): 680-685.Polarization of fluorescence measurements on hen's egg white lysozyme labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride have been used to detect the formation of a soluble complex of this protein with thyroglobulins from bovine, normal human, and goitrous human thyroids. The binding of the labeled lysozyme to thyroglobulin is accompanied by a 40% increase in the quantum yield, a blue shift of 14 nm in the emission maximum, and a lengthening of the fluorescence lifetime of the 1-dimethylaminonaphthalene-5-sulfonyl moiety from 6.7 to 8.2 nsec. A statistical dissociation constant of 1.5 × 10-6 m at pH 8.0 and evident cooperativity of binding indicated by a Hill exponent of 3 to 4 were observed. Assuming that interlysozyme bonds occur when 2 or more lysozyme molecules are bound contiguously on thyroglobulin, a free energy of lysozyme-lysozyme interaction of about 1 kg cal per pair accounts well for the observed cooperativity.
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Addition of chemical and osmotic free energies through negative interaction of protein-bound ligands.
Proc Natl Acad Sci USA. 1972; 69(10): 3000-3003. PMCID: PMC389694Simple free energy considerations show that an effector ligand, Y, bound to a protein can modify the standard free energy change of the reaction P -X + D- R = P - R + D - X, where P is a protein, D - R a small molecule, and X a group in the protein that interacts negatively with Y. By consideration of a three-compartment system containing the protein, an X-acceptor (A -R), an X-donor (D - X), and the ligand Y present at two different concentrations in the inner and outer compartments, it is shown that the protein can perform under steady-state conditions the addition of the free energy in the Y concentration gradient to that of the chemical reaction A - X + D - R = A - R + D -X. This free energy addition can effectively generate a high energy X-donor from a low energy ... [truncated at 150 words]
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Uses of fluorescence in biophysics: some recent developments.
Annu Rev Biophys Bioeng. 1972; 1: 553-570.The use of fluorescence emission to study various properties of biological systems has undergone rapid development in the past fifteen years so that it is already next to impossible for any one person to keep up with the vast amount of information presented in the many published studies. I have not, therefore, attempted a thorough survey of the field but have concentrated attention on a few points on which advances in the last five years have seemed to me particularly impressive and where fuller development promises to yield information of singular biological interest....
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Strong binding of hydrophobic anions by bovine serum albumin peptides covalently linked to lysozyme.
Biochemistry. 1971; 10(24): 4492-4495.The small-peptide fraction, derived from a limited chymotryptic digestion of bovine serum albumin, and representing nearly 2 0 z of the total protein weight, contains the strong hydrophobic anion binding sites of intact albumin. When these small peptides are covalently attached to lysozyme, used as a macromolecular support, binding of 1-anilinonaphthalene-8-sulfonate is only half an order of magnitude weaker than binding by native bovine serum albumin. In contrast, a lysozyme control, lysozyme derivatives containing peptides obtained from the macromolecular fraction of digested albumin, and the free small-peptide fraction have very low binding affinities for the same ligand.
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Theory of fluorescence depolarization by anisotropic brownian rotations: discontinuous distribution approach.
J Chem Phys. 1971; 55(5): 2399-2407.In this paper, we generalize to molecules devoid of symmetry the phenomenological treatment of depolarization of the fluorescence by the Brownian rotations of a spherical molecule due to Spencer and Weber. There are six ways in which a system of orthogonal coordinates rigidly bound to the molecule may be made to coincide with the laboratory coordinates. The transitions between these permissible orientations gives rise to a system of six differential equations of transport, the solution of which entails that of a differential equation of sixth order. However, because of the equivalence of some of the rotations as regards the depolarizing effects, on account of the symmetry conditions, the solution of a differential equation of third order is only necessary. Consequently, after an instantaneous light pulse, a polarized component of the fluorescence will show a maximum of three exponential decays and the difference of parallel and perpendicular components a maximum of ... [truncated at 150 words]
Weber G, Borris DP, de Robertis E, Barrentes FJ, Torre JLL, de Carlin MCL.
The use of a cholinergic fluorescent probe for the study of the receptor proteolipid.
Mol Pharmacol. 1971; 7(5): 530-537.A trimethylammonium derivative, 1-dimethylaminonaphthalene-5-sulfonamidoethyl-trimethylammonium perchlorate, was synthesized and used as a fluorescent probe for the study of the receptor proteolipid extracted from the electric organ of Electrophorus electricus.
A special technique based on the preferred partition coefficient of the proteolipid for organic solvents and of the trimethylammonium derivative for water was developed. Such a method permits time accurate and rapid titration of the proteolipid with the cholinergic fluorescent probe and also the study of the action of other drugs competing for the binding sites.
A cooperative type of interaction between the fluorescent trimethylammonium derivative and the receptor proteolipid was observed, and the competition of acetylcholine, dimethyl-d-tubocurarine, and decamethonium for the binding sites of the fluorescent probe was demonstrated.
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Presence of arginine residues at the strong, hydrophobic anion binding sites of bovine serum albumin.
Biochemistry. 1971; 10(8): 1335-1339.Derivatives of bovine serum albumin were prepared by partial chemical modification of the native protein with acefic anhydride, formaldehyde, and glyoxal. Rotational relaxation time, sedimentation velocity coefficient, and pH dependence of fluorescence polarization measurements indicate that the overall tertiary structure of the derivatives is the same as that of bovine serum albumin in the pH range from 4.5 to 10. However, the formaldehyde- and glyoxal-treated albumins with 30 and 80% modified arginine residues, respectively, have a binding affinity for l-anilinonapthalene-8-sulfonate almost two orders of magnitude lower than the acetylated and native albumins. The results suggest that there are arginine residues at or close to the strong hydrophobic anion binding sites of bovine serum albumin.
Shinitzky M, Dianoux AC, Gitler C, Weber G.
Microviscosity and order in the hydrocarbon region of micelles and membranes determined with fluorescent probes. I. Synthetic micelles.
Biochemistry. 1971; 10(11): 2106-2113.Thee viscosity in micelle interiors, termed here as microviscosity, was derived from an adequate comparison of the degree of fluorescence depolarization of perylene or 2-methylanthracene when dissolved in the tested micelles and in American white oil U.S.P.35. The latter was used as a reference system of known viscosities. In the series studied, lauryltrimethylammonium bromide, myristyltrimethylammonium bromide, cetyltrimethylammonium bromide (CTABr), and stearyldimethylbenzylammonium bromide, the determined microviscosities at 27° are all in the range of 17-50 cP. The change in microviscosity with temperature in this series was found to follow a simple exponential form with an activation energy in the range of 6.1-9.6 kcal mole^-1. Added salts affected only slightly the microviscosity values. Mixed micelles of perylene-labeled CTABr with cetyl alcohol or cholesterol and with sodium 1-hexadecanesulfonate, were used to test the effect of charge isolation and charge neutralization on the fluidity of the micelle interior. The microviscosity of these mixed micelles ... [truncated at 150 words]
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The binding of small ligands by proteins.
3rd International Congress on Hormonal Steroids, Hamburg, 7-12 September 1970.
Hormonal Steroids (International Congress Series). By V Hector, T James, and L Martini (Editors). Elsevier Science, pp. 58-65, 1971. ISBN: 9789021901442
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Limited digestion of citraconylated bovine serum albumin with alpha-chymotrypsin.
Biochemistry. 1970; 9(26): 5092-5099.Citraconyl bovine serum albumin was digested with a-chymotrypsin until eight to nine amide bonds had been broken per protein molecule. Under the conditions of the digestion (i.e., pH 7.7, water solution, 23°) the citraconylated protein, which had nearly 70% of its e-amino groups modified by citraconic anhydride, existed in an expanded conformation very similar to that of serum albumin in acid (Jonas, A., and Weber, G. (1970), Biochemistry 9, 4729). The digest was fractionated on ion-exchange and gel filtration columns, into a small peptide fraction and several macromolecular fragments. These chymotryptic fractions were subsequently studied as to their average molecular weights, rotational relaxation times, amino acid composition, fluorescence spectra, and anion binding properties. Molecular weights and rotational relaxation times for the five macromolecular fractions ranged from 35,000 to 8000 and from 33 to 6 nsec, respectively. The per cent amino acid compositions deviated significantly in polar residue content from that ... [truncated at 150 words]
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Partial modification of bovine serum albumin with dicarboxylic anhydrides. Physical properties of modified species.
Biochemistry. 1970; 9(24): 4729-4735.Bovine serum albumin was modified chemically with succinic, maleic, and citraconic anhydrides, and the properties of the derivatives were studied as to the nature and extent of the structural changes in the protein. Fluorescence polarization, rotational relaxation time, and sedimentation velocity measurements, as well as ionic strength, nature of salt, and pH dependence of fluorescence polarization experiments, led to the specification of conditions (e.g., pH 7.0, water solution, 20°, for a 65 % modified sample) under which the bovine serum albumin derivatives were shown to exist in an expanded form, very similar in its physical properties to bovix serum albumin expanded by electrostatic repulsion at pH 2. Modification of bovine serum albumin with citraconic anhydride was shown to be reversible; the citraconyl groups were easily removed under mild acidic conditions, without affecting the physical properties of the protein in relation to a control bovine serum albumin sample. The expansion of ... [truncated at 150 words]
Anderson SR, Brunore M, Weber G.
Fluorescence studies of Aplysia and sperm whale apomyoglobins.
Biochemistry. 1970; 9(24): 4723-4729.We have investigated some of the molecular properties of the globins prepared from Aplysia myoglobin, sperm whale myoglobin, and human hemoglobin using fluorescence and fluorescence polarization methods. The study of the intrinsic fluorescence of the apomyoglobins shows definite differences between the two proteins. The fluorescence spectrum of Aplysia apomyoglobin clearly reveals two different tryptophan residues with emission maxima at ca. 330 and 355 nm. These wavelengths correspond, respectively, to the maxima of tryptophan in nonpolar and in aqueous solvents. The fluorescence polarization spectrum of Aplysia apomyoglobin demonstrates localized rotational freedom expected for a mobile tryptophan side chain in contact with the water. In contrast, the two tryptophan side chains of sperm whale apomyoglobin are rigidly bound in similar nonpolar regions within the protein matrix. The average lifetime of the excited state is in the range 2.8-2.9 nsec for both apomyoglobins. The fluorescence emission maximum of adsorbed l-anilino-8-naphthalenesulfonate occurs at 478 ... [truncated at 150 words]
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Failure of energy transfer between identical aromatic molecules on excitation at the long wave edge of the absorption spectrum.
Proc Natl Acad Sci USA. 1970; 65(4): 823-830. PMCID: PMC282989Electronic energy transfer among identical molecules has been followed by the depolarization of the fluorescence in concentrated solutions as well as in dimers, polymers, and micelle systems. In the many aromatic fluorophores examined, unlike a few nonaromatic ones, transfer is much decreased or altogether undetectable on excitation at the red edge of the absorption spectrum. The phenomenon is not due to the transfer taking place during a small fraction of the total fluorescence lifetime, nor is it explainable by a decrease in overlap of absorption and emission upon edge excitation.
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Influence of brownian rotations and energy transfer upon the measurements of fluorescence lifetime.
J Chem Phys. 1970; 52(4): 1654-1663.The depolarization of the fluorescence of solutions by either Brownian rotations or intermolecular energy transfer may be simply described by a system of first-order linear differential equations containing as only parameters the rate of fluorescence emission and the rate of transport of the excitation from one orthogonal component of the emission to another. The steady-state solution has the form of Perrin's equation describing the depolarization by Brownian rotations, and the time-dependent depolarization following a unit light impulse is that originally described by Jablonski. The solution for sinusoidal excitation is novel in that: 1. It shows the difference in lifetime between the polarized components of the emission to be a sensitive function of the ratio of the modulation frequency omega to the emission rate lambda. For omega/lambda > 1 the difference between the polarized lifetimes may become many times greater than that observed after a unit light impulse. 2. It permits ... [truncated at 150 words]
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Oxygen quenching of pyrenebutyric acid fluorescence in water. A dynamic probe of the microenvironment.
Biochemistry. 1970; 9(3): 464-473.Solutions of pyrenebutyrate have fluorescence lifetimes of 135 nsec in deoxygenated water and 100 nsec, and a corresponding diminished yield in air-equilibrated water. The oxygen quenching of the fluorescence is shown by means of measurements of lifetime and relative yield of fluorescence to be a diffusion-controlled phenomenon in which virtually every collision is effective. Adsorbates or chemical conjugates of pyrenebutyric acid with bovine albumin are not affected by changing the concentrations of 0, in the medium, while in concentrated urea the quenching is markedly increased. Observations of a similar kind have been carried out in polylysine conjugates and apohemoglobin adsorbates. The lifetime in the absence of oxygen (τ0) the systems studied varied from 100 to 205 nsec. From observations of glycerol-water mixtures it is suggested that τ0 may depend upon the polarity of the environment. The observations reported here show that pyrenebutyric acid may be used to determine the accessibility ... [truncated at 150 words]
Scott TG, Spencer RD, Leonard NJ, Weber G.
Emission properties of NADH: studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, and simplified synthetic models.
J Am Chem Soc. 1970; 92(3): 687-695.The fluorescence lifetimes T and quantum efficiencies q of NADH, AcPyADH, and of the model compounds Ad-Ca-NicH and Ad-Ce-NicH have been measured in water and in 1,2-propanediol solution in the temperature range 0-30”. For Ad-Ca-NicH, NADH, and Ad-C6-NicH at 25” in water the absolute quantum efficiencies are 0.035, 0.019, and 0.017, respectively, to a precision of a few per cent, and the lifetimes are 0.70, 0.40, and 0.28 f 0.03 to 0.05 nsec The contribution of the ground to lowest singlet transition to the absorption spectrum has been evaluated by fluorescence polarization observations, and from these and the molecular fluorescence spectra, emissive lifetimes re have been calculated by the equation of Strickler and Berg. For NADH and AcPyADH under various conditions, the relation τ = τeq is followed rigorously over a thirtyfold change in quantum efficiency. The absolute efficiency of quinine sulfate measured either by comparison with these derivatives or ... [truncated at 150 words]
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Molecular complexes of biological interest.
4th International Congress on Pharmacology, July 14-18, 1969 in Basel, Switzerland.
Proceedings of the Fourth International Congress on Pharmacology (Vol. 3). By R Eigenmann (Editors). Schwabe, pp. 180-191, 1970.
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Fluorescence methods in the study of protein conformation.
Great Lakes Regional Meeting
of the American Chemical Society, Chicago, 1969.
Spectroscopic approaches to biomolecular conformation. By DW Urry (Editors). American Medical Association, pp. 23-31, 1970.
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Fluorescence polarization of pyrenebutyric-bovine serum albumin and pyrenebutyric-human macroglobulin conjugates.
J Biol Chem. 1969; 244(23): 6309-6315.Pyrenebutyric acid when conjugated with proteins has a long fluorescence lifetime in the range of 100 nsec as measured both directly and indirectly. The fluorescence life-times as measured indirectly by the fluorescence depolarization of pyrenebutyric-bovine serum albumin conjugates appears to be a linear function of quantum yield. This dependence was used to predict the lifetimes of the other protein conjugates. The rotational relaxation time of the conjugate of rabbit immunoglobulin G agrees with other determinations with a different chromophore. Conjugates were prepared with human immunoglobulin M, and the measured rotational relaxation times of the conjugate before and after reduction and alkylation correlated with the molecular weights of the 2 molecules. In addition, the reduction of the conjugate is followed, and evidence is presented supporting the concept that a greatly disordered conformation is an intermediate.
Rawitch AB, Hudson EN, Weber G.
The rotational diffusion of thyroglobulin. A study of thyroglobulin conjugated with pyrenebutyrate and with dimethyl-aminonaphthalenesulfonate.
J Biol Chem. 1969; 244(23): 6543-6547.Conjugates of thyroglobulin with dimethylaminonaphthalenesulfonate and pyrenebutyrate have been prepared, and their fluorescence has been utilized to study the rotational diffusion of this large glycoprotein. A method of activating the pyrenebutyrate moiety by conversion to the mixed sulfuric anhydride is described. Labeling with this reagent may be carried out in aqueous solution containing less than 1% organic solvent and in periods of under 1 hour. Isothermal polarization measurements on both human and bovine thyroglobulins indicate a rotational relaxation time near 1.5 µsec at 25° in water and suggest a rigid structure which deviates significantly from an anhydrous sphere. Measurements of the polarization of fluorescence of dimethylaminonaphthalenesulfonate conjugates at various temperatures indicate the existence of thermally activated rotations of the fluorescent dye moiety which can result in the significantly shorter apparent rotational relaxation times previously reported. An Arrhenius plot of limiting polarization data indicates an activation energy barrier of 0.59 kcal ... [truncated at 150 words]
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Dimer formation from 1-anilino-8-naphthalenesulfonate catalyzed by bovine serum albumin. A new fluorescent molecule with exceptional binding properties.
Biochemistry. 1969; 8(10): 3915-3920.At pH values around 2, nitrite induces a series of chemical changes in 1-anilino-8-naphthalenesulfonate. From the resulting mixture of products a compound has been isolated,
the spectroscopic properties of which are very similar to the parent compound, but with an affinity for bovine serum albumin nearly two orders of magnitude greater than that of the latter. The number of strong binding sites displaying fluorescence enhancement is two. If albumin is present during the formation of the compound, the yield, which otherwise amounts to only 1-2%, approaches l00%, i.e., the protein appears to behave in an enzyme-like fashion. Three different preparative procedures are described. Experimental evidence is given to support the conclusion that the new molecule is a dimer of 1-anilino-8-naphthalenesulfonate.
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Measurements of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer.
Electronic Aspects of Biochemistry (Annals of the New York Academy of Sciences, Vol. 158). pp. 361-376, 1969. Introduction: The methods for the direct determination of fluorescence lifetimes may be discussed by considering each single fluorescent species as a detector in which the response to an infinitely narrow pulse of light decays in time, t, according to the exponential law I(t) = I(0) × exp - (kt). From the relations of the exciting light with the light emitted by such exponential detector, the rate constant k or its reciprocal τ, the lifetime of the excited state may be determined...
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The effects of energy transfer and rotational diffusion upon the fluorescence polarization of macromolecules.
Biochemistry. 1969; 8(1): 361-371.The rotational relaxation time of a macromolecule computed from data of fluorescence polarization is an average of the principal relaxation times of the associated hydrodynamic ellipsoid weighted according to the orientation of the transition moments in absorption and emission with respect to the ellipsoid's axes. Memming's equations, describing the fluorescence polarization of these cases, are transformed so as to express the polarization as an explicit function of the angle θ between the transition moments, and the angle γ between the plane containing them and the equator of the ellipsoid. This transformation is particularly suitable to enumerate the cases arising when an aromatic ligand with π -> π* electronic transitions in absorption and emission is bound to a macromolecule with preferential orientation. The effects of energy transfer among bound ligands may also be easily visualized and calculated. We have made calculations for a typical prolate ellipsoid of axial ratio 4, varying ... [truncated at 150 words]
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Fluorescence polarization of the complexes of 1-anilino-8-naphthalenesulfonate with bovine serum albumin. Evidence for preferential orientation of the ligand.
Biochemistry. 1969; 8: 371-375.The rotational relaxation time <ρ> of the complexes of 1-anilino-8-naphthalenesulfonate with
bovine serum albumin has been calculated from independent measurements of fluorescence polarization and lifetime for different values of n, the average number of 1-anilino-8-naphthalenesulfonate molecules bound per molecule of bovine serum albumin. It increases from
105 ± 3 nsec at n =1 to 128 ± 3 nsec at n = 5 when the wavelength of excitation is 366 nm, but remains constant at 105 nsec for all values of d when excitation is at 436 nm. Depolarization by energy transfer in both the bovine serum 1-anilino-8-naphthalenesulfonate adsorbates and concentrated solutions of 1-anilino-8-naphthalenesulfonate in propylene glycol is wavelength dependent, transfer failing in both cases upon excitation at 436 nm. Energy transfer is therefore the origin of the apparent increase in <ρ> with n. Analysis of the results according to the preceding paper shows that a model of preferential orientation of the 1-anilino-8-naphthalenesulfonate ... [truncated at 150 words]
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Fluorescence methods in kinetic studies.
Fast Reactions (Methods in Enzymology, Vol. 16). By K Kustin (Editors). Academic Press, pp. 380-393, 1969. ISBN: 012181873XIntroduction: The absorption of light by organic molecules results in the appearance of an excited state characterized by structural and functional properties often very different from those of the ground state. Although it is possible to reach upon absorption any of several excited states of the singlet manifold, redistribution of the energy among the degrees of freedom of the excited molecule, as well as energy exchange with the surrounding molecules, results in appreciable population of only the lowest excited singlet or triplet...
Spencer RD, Vaughan WM, Weber G.
Dependence of fluorescence lifetimes upon the excitation and fluorescence spectra.
International Conference on Molecular Luminescence, Loyola University, Chicago, August 1968.
Molecular Luminescence. By EC Lim (Editors). Benjamin, New York, pp. 607-629, 1969.
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The reversible acid dissociation and hybridization of lactic dehydrogenase.
Arch Biochem Biophys. 1966; 116(1): 207-223.Polarization spectra of the intrinsic fuorescence of lactic dehydrogenase (LDH) demonstrate a reversible structural transition in acidic solutions. The pH region of this transition varies for different LDH's. The transition is marked by loss of coenzyme binding and catalytic activity, enhanced ability to bind 1-anilinonaphthalene-8-sulfonate, and a decrease in rotational relaxation time from 188 nseconds at pH 7.1 to 77 nseconds at pH 2.5. Renaturation by dilution into neutral solution leads
to a recovery of more than 90% LDH with catalytic and physical properties identical to those of the untreated enzyme. Hybrids of beef heart LDH X beef muscle LDH and of beef heart LDH X chicken heart LDH have been prepared by neutralization of solutions of LDH incubated in acid. Our results indicate that a reversible dissociation, either partial or complete, is the basis of hybridization.
Measurements of fluorescence polarization, coenzyme binding, and ease of hybridization demonstrate that the hybrids ... [truncated at 150 words]
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The effect of magnesium on some physical properties of yeast enolase.
J Biol Chem. 1966; 241(11): 2550-2557.It is shown by sedimentation velocity and polarization of fluorescence measurements that yeast enolase in solution becomes more compact on addition of magnesium. This is associated with changes in absorption, fluorescence emission, and fluorescence-polarization spectra, as well as in the thermal stability of the enzyme. Evidence is presented that this structural change is produced by the binding of 1 mole of metal per mole of protein. The dissociation constant, as determined by fluorimetric titrations, is 1.8 ± 0.5 10-6 M.
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Construction and performance of a fluorescence polarization spectrophotometer.
J Biol Chem. 1966; 241(11): 2558-2561.The construction and performance of a fluorescence polarization spectrophotometer is described, the parts of which are mostly commercially available items. The instrument uses two photomultipliers to detect separately each polarized component of the fluorescent light. The ratio of the components is plotted continuously by means of a ratio recorder while the wave length of the exciting light is changed at uniform speed. A servomechanism keeps the slits to the minimum value compatible with the sensitivity of the instrument. We obtained maximum resolution on the excitation side by allowing the broadest possible band of fluorescence, selected by broad band or low pass optical filters, to reach the photomultipliers. The use of fluorescence polarization spectrophotometry in investigation of proteins is discussed.
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Cooperative binding by bovine serum albumin. I. the binding of 1-anilino-8-naphthalenesulfonate: Fluorimetric titrations.
Biochemistry. 1966; 5(6): 1893-1899.The binding of anilinonaphthalenesulfonate by bovine serum albumin at various values of pH, temperature, and ionic strength has been studied by a fluorometric technique. Five moles of anilinonaphthalenesulfonate was bound/mole of serum albumin over the pH range 10-5.0. The equilibrium concentrations of free ligand, free protein, and complex were determined for values of the probability of binding from 0.05 to 0.95. The more complete titration curves obtained contained some 20 binary units of information. Abrupt changes in the apparent reaction order, similar to those demonstrated in the equilibrium of reduced diphosphopyridine nucleotide (DPNH) and beef muscle lactate dehydrogenase, were observed at pH 7, and a continuous increase in the reaction order with probability of binding was seen at pH 5. The results of pH 7, and probably also those at pH 5.0, are not expressible by an equation of Adair's type. We believe that, as in the case of lactate ... [truncated at 150 words]
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Cooperative effects in binding by bovine serum albumin. II. the binding of 1-anilino-8-naphthalene-sulfonate: Polarization of the ligand fluorescence and quenching of the protein fluorescence.
Biochemistry. 1966; 5(6): 1900-1907.The decrease in the polarization of the fluorescence of anilinonaphthalenesulfonate adsorbed upon bovine serum albumin with the average number bound is found to be due solely to electronic energy transfer among the ligand molecules. A descriptive theory of this phenomenon is developed using two simplifying assumptions : (1) random distribution of the ligand molecules among the protein binding sites. (2) A single transfer of the excited state is responsible for the depolarization. Under these assumptions, a "system of equivalent oscillators" may be defined which best fits the experimental data. The equivalent system for the albumin-anilinonaphthalenesulfonate case is
one in which the average distance between a pair of binding sites is 21 Å and the average angle between two emission oscillators is 33°. The polarization data show the existence of cooperative features in the binding at pH 5 by comparison with that at pH 7, a phenomenon already seen in the titration ... [truncated at 150 words]
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Polarization of the fluorescence of solutions.
Fluorescence and Phosphorescence Analysis. By DM Hercules (Editors). Interscience Publishers, pp. 217-240, 1966.
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A low intensity permanent liquid light standard activated by radioactivity.
Photochem Photobiol. 1965; 4(6): 1049-1050.The total radiant flux from a solution containing a radioactive compound, hexadecane-1-14C, plus an appropriate scintillator, has been determined. The procedure used involves comparison of its luminescent emission with the light scattered by glycogen illuminated with a monochromatic homogeneous light beam of known photon flux. These standards are particular convenient for use with chemiluminescent and bioluminescent reactions, since the geometry of the emission may be duplicated exactly.
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Multiplicity of binding: range of validity and practical test of Adair's equation.
Biochemistry. 1965; 4(10): 1942-1947.In this paper we discuss the theoretical aspects of binding necessary for the interpretation of the experimental results to be described in the second paper. Frequent reference will also be made to a recent publication by one of us (Weber, G., 1965, in Molecular Biophysics, Pullman, B., and Weissbluth, M., eds., New York, Academic) where the general problem of binding of small molecules to proteins is discussed in detail. To begin with we consider the information content of binding experiments and the form of presentation of the experimental data. Next we examine the problems of cooperative binding: in attempting a description of cooperative binding a distinction must be made between those cases in which the dissociation constants of the protein-ligand complexes are independent of the degree of saturation of the system and those in which they are functions of it. We shall demonstrate that binding of an arbitrary number of ... [truncated at 150 words]
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Multiplicity of binding by lactate dehydrogenases.
Biochemistry. 1965; 4: 1948-1957.We have studied the binding of nicotinamideadenine dinucleotide (NADH) by the five forms of beef lactate dehydrogenase using fluorescence methods. Through the use of a dilution procedure in which the ratio of coenzyme to enzyme is kept constant and nearly stoichiometric, we have been able to evaluate the equilibrium concentrations of free protein, free ligand, and complex over nearly 95% of the titration range. Ten to twenty binary units of information were obtained in the different cases. Previous work covered only 30% of the titration curve and contained no more than three binary units of information. The different lactate dehydrogenases bind 4 moles of coenzyme/l33,000-140,000 of protein. At pH 7.4, 0.05 M phosphate buffer,beef heart lactate dehydrogenase was found to have four independent, equal sites with K = 3.9 ± 0.4 × 10-7 M.
Titrations of beef muscle lactate dehydrogenase showed marked changes in the order of the binding reaction ... [truncated at 150 words]
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Polarization of the fluorescence of solutions subjected to an electric field.
J Chem Phys. 1965; 43(2): 521-524.The dipole moments of fluorescent molecules in nonpolar solvents in the lowest singlet excited state has been previously calculated from observations of the increase in fluorescence polarization when the molecules are subjected to an electric field on the assumption that the Langevin distribution prevails at the time of the emission. By the use of Benoit's theory of the rotational diffusion of a dipole placed in an electric field, and under conditions of isotropic depolarization, an equation is obtained which allows the calculation of the excited dipole moment when the equilibrium distribution is not reached during the excited state.
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Interaction of proteins with radiation.
The Proteins (Vol. III, 2nd edition). By H Neurath (Editors). Academic Press, New York, pp. 445-521, 1965.
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The binding of small molecules to proteins.
Molecular Biophysics: Proceedings of an international summer school held in Squaw Valley, California, August 17-28, 1964. By B Pullman and M Weissbluth (Editors). Academic Press, New York, pp. 369-396, 1965. INTRODUCTION: The interaction of small molecules with proteins has been the subject of extensive research, both theoretical and experimental. Interest in this, subject has followed the recognition that the physicochemical basis of biological specificity ought to be exhibited in its utmost simplicity in the interaction of proteins and small ligands. I do not intend to deal here either with the forces responsible for such interactions or with their ultimately expected relation to biological specificity: Before each of these two complex tasks is contemplated in any particular case, one ought to attempt to describe as completely and succinctly as possible the system in question. In this task, we should seek to avoid presuppositions as to the simplicity or multiplicity of the process, as to the homogeneous or heterogeneous character of the population involved, or as to the number of the parameters required for its description. This may be achieved by introducing ... [truncated at 150 words]
Gibson QH, Hastings JW, Weber G, Duane W, Massa J.
The interaction of flavin mononucleotide with an enzyme system from Photobacterium fischeri.
Flavins and Flavoproteins (BBA Library, Vol. 8). By EC Slater (Editors). Elsevier Publishing Co, pp. 341-359, 1965. ISBN: 9780444405432-
Intramolecular complexes of Flavins.
Flavins and Flavoproteins (BBA Library, Vol. 8). By EC Slater (Editors). Elsevier Publishing Co, pp. 15-21, 1965. ISBN: 9780444405432
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Fragmentation of bovine serum albumin by pepsin. I. The origin of the acid expansion of the albumin molecule.
J Biol Chem. 1964; 239(5): 1415-1423.
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Fragmentation of bovine serum albumin by pepsin. II. Isolation, amino acid composition and physical properties of the fragments.
J Biol Chem. 1964; 239(5): 1424-1431.In the previous paper, the change in physical properties of solutions of bovine serum albumin and of a conjugate of bovine serum albumin with 1-dimethylaminonaphthalene-5-sulfochloride subjected to a short peptic digestion at pH 3.0 were examined. It was concluded that the action of pepsin resulted in the liberation of a small number of units, probably globular in character, that were linked together in the native protein by the continuity of the peptide chain. The present paper describes the isolation of the fragments of digestion and the results of measurements of their amino acid composition, sedimentation, rotational and translational diffusion, absorption spectrum, and fluorescence spectrum.
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Proton-transfer effects in the quenching of fluorescence of tyrosine copolymers.
A symposium held at Stanford University, California, March 25-29, 1963.
Quantum Aspects of Polypeptides and Polynucleotides (Vol. 13). By M Weissbluth (Editors). Interscience Publishers Inc, pp. 333-341, 1964. ISBN: 9780470932735The quantum yield of the fluorescence of tyrosine in a variety of proteins, both containing and free from tryptophan, has been found uniformly low, in most cases in the range 0.015--0.04, as compared with a value of 0.22 in phenol, cresol, and 0,21 in free tyrosine, Competitive processes capable of reducing the quantum yield of the fiuorescence by internal conversion of the electronic energy can arise under a variety of circumstances, so that the experimental approach to the protein problem through the use of model systems of increasing complexity seems indispensable.
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Enumeration of components in complex systems by fluorescence spectrophotometry.
Nature. 1961; 190(4770): 27-29.The constancy of the spectral distribution of the fluorescence for a given substance excited with light of different wave-lengths permits the development of a simple...
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pH dependence of the fluorescence yield of tyrosine copolymers.
404th Meeting of the Biochemical Society, London, March 27-29, 1961.
Biochem J. 1961; 79(3): 29, 19.Two copolyrnors, one (GT) containing 4 % of tyrosine and 96 % of glutamic acid, another (GLT) containing 4 % of tyrosine, 58 % of glutamic acid and 36 % of lysine, were examined. The fluorescence spectra of both copolymers at all pH values were indistinguishable from that of tyrosine (emission maximum at 303 ± 1 mμ).
GT had a quantum yield q = 0.02 at pH 9-6.5, both in salt-free solution and in M-NaCl. Below pH 5 the yield rose reaching q = 0.13 without salt and 0.175 in M-NaCl, at pH 2. The behaviour of GT after O-methylation of the tyrosines with ditnethyl sulphate was similar in the acid region but in the range of pH 4.5-11 the yield was over three times higher (q = 0.069). Prom these observations and the quenching of the fluorescence of phenol and anisole by carboxylic acids (White, 1960) it is concluded ... [truncated at 150 words]
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Tyrosine fluorescence in the albumins.
404th Meeting of the Biochemical Society, London, March 27-29, 1961.
Biochem J. 1961; 79(3): 29-30, 20.Examination of the fluorescence spectrum of proteins containing both tyrosine and tryptophan has has shown no indication of tyrosine fluorescence (Teale, 1960). The use of the matrix method in the analysis of the spectrofluorometric data (Weber, 1961), shows the presence of two components and two components alone, in the fluorescence spectrum of human-serum albumin (HSA), bovineserum albumin (BSA) and ovalbumin. From difference fluorescence spectra the second component may be shown to have a maximum of emission at 305 ± 3 mμ and a quantum yield of 0.018-0.022 in the three intact proteins. The second component is excited with a lesser efficiency than the main tryptophan fluorescence by wavelengths shorter or longer than 280 mμ and from this fact, and the emission spectrum, it may be identified as tyrosine. In solutions in 8M-urea the yield of the tyrosine fluorescence increases to 0.035 in HSA and 0.030 in BSA, while the fluorescence ... [truncated at 150 words]
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Excited states of proteins.
Symposium on Light and Life. March 28-31, 1960. Johns Hopkins University, Baltimore.
Light and Life. By HB Glass and WD McElroy (Editors). Johns Hopkins Press, pp. 82-106, 1961. ISBN: 9780801804113
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Energy transfer with special reference to biological systems.
Nature. 1959; 184(4687): 688-709....energy may be transferred from one atom or molecule to another have become a prominent theme of chemical kinetics, photochemistry and radiation physics as well as of many...
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Ultraviolet fluorescence of proteins.
Proceedings of the Biochemical Society, London, 1959.
Biochem J. 1959; 72(3): 15.Tyrosine, tryptophan and phenylalanine have characteristic fluorescence in water solution (Teale & Weber, 1957). In the protein molecule the environment of the aromatic residues is modified in several ways: the surrounding medium is less polarizable than water, rotational freedom is restricted and adjacent groups of the polypeptide framework may take part in specific quenching processes. In addition, the average distance among the aromatic residues is about 10Å, a distance at which energy transfer is known to take place in other systems (Förster, 1951). These possibilities have been examined in detail in solutions of twenty-six globular proteins. In all cases phenylalanine fluorescence was absent and that of tyrosine was observed only in the absence of tryptophan. In ribonuclease, insulin and zein tyrosine fluorescence with quantum yield of 0.03 on the average was present, while two trypsin inhibitors containing only tyrosine were non-fluorescent. Tryptophan fluorescence, and this fluorescence alone, was observed whenever ... [truncated at 150 words]
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Polarization of the ultraviolet fluorescence and electronic energy transfer in proteins.
Proceedings of the Biochemical Society, London, 1959.
Biochem J. 1959; 72(3): 15.The fluorescence of solutions of phenol, cresol and indole in propylene glycol at -70° in layers of 20-30 µ thickness showed concentration depolarization (Förster, 1951; Weber, 1954) over the range 0.01-0.1 M. The characteristic distances R for which the chances of transfer and emission are equal is for both phenol and indole 17Å. Transfer in indole is only observed when the wavelength of excitation is shorter than 300 mµ. Even 0.4M-indole solutions show strong polarization when excited with 310 mµ light. In mixtures of phenol and indole studied under the same conditions polarization and fluorescence spectral measurements show transfer from phenol to indole with R = 20Å. Proteins that do not contain tryptophan (zein, insulin, ribonuclease), dissolved in 50% propylene glycolwater mixtures at -70° and excited with light of 275-290 mµ, wavelength show polarizations of 0.10-0.14, while cresol or phenol in 10-4M solution show in the same conditions a polarization ... [truncated at 150 words]
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Nature of the conformational changes of albumin in acid.
Proceedings of the Biochemical Society, London, 1959.
Biochem J. 1959; 72: 74.Serum albumin is known to change reversibly its hydrodynamic and optical properties in the pH range 4-2. Rotational and translational diffusion studies and low-angle X-ray scattering (Luzzati, Witz & Nicolaeiff, 1961) are all in accord with a model of compact units linked by a flexible ...
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Electronic energy transfer in haem proteins.
Discuss Faraday Soc. 1959; 27: 134-141.The fluorescence efficiency of the tryptophan fluorescence of haem proteins is very small, in most cases smaller than 0.002, while after removal of the haem is around 0.2. The fluorescence efficiency of crystallizable conjugates of the haem proteins with dimethylamino naphthalene sulphonyl chloride (DNS) before and after removal of the haem has been determined. On the assumption of the uniform random distribution of the labels over the spherical protein surface, the one-haem proteins myoglobin and horse-radish peroxidase give as the distance R at which the probability of transfer of the excited state to the haem equals the probability of emission by the DNS residue, a value of 42 Å, to be compared with 58 Å calculated by the theory of Forster. In the four-haem proteins haemoglobin and catalase the values of R are 65 8, and 66 8, if the haem sare supposed to be crowding together at one place ... [truncated at 150 words]
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Kinetic studies on rabbit-muscle myokinase.
Biochem J. 1959; 73(3): 473-485. PMCID: PMC1197084Myokinase (adenylate kinase) catalyses the reaction:
2 Adenosine diphosphate = Adenosine triphosphate + adenosine monophosphate (1)
Highly purified preparations of this enzyme have been obtained from rabbit muscle by Noda & Kuby (1956, 1957a, b) and Callaghan (1956, 1957a, b), and the preparation of the last-named author, which appears to contain a single protein component, has been used for the present studies of the mechanism of myokinase action. Several possible mechanisms by which myokinase might catalyse transphosphorylation among adenosine nucleotides were considered by Whittam, Bartley & Weber (1955). The enzyme may accept the terminal phosphate of adenosine triphosphate (ATP) or adenosine diphosphate (ADP), thus forming an intermediate phosphorylated protein by the reactions...
