Gregorio Weber - Research Accomplishments

Summary

Gregorio Weber's career had two distinct periods.  In the first, from 1947 to 1975, he contributed to the development of fluorescence instrumentation and to the theory and procedures of determination of quantum efficiency, fluorescence polarization, fluorescence spectrum, and also to the analytical determination of the number and character of the components of composite fluorescence.  The latter has been an indispensable tool in the examination of natural systems where the origin of the fluorescence is always heterogeneous.  The fluorescence polarization techniques developed by Weber have been applied to numerous problems of clinical investigation, to the diagnostic determination of drugs and metabolites in the blood and to the sequence of amino acids in proteins and of bases in the nucleic acids.  Weber devoted a great deal of time and effort to improving the determination of fluorescence lifetimes and created the "cross correlation" technique of phase fluorometry that forms the current basis of the phase measurements of lifetimes. In recent times it has lead to applications in microscopy and even to the macroscopic imaging of tissues (E. Gratton and coworkers).  From 1965 onwards, initially in collaboration with H.G. Drickamer, he applied high pressure fluorescence spectroscopy to the study of molecular complexes and proteins.  These observations confirmed the power of the fluorescence techniques to resolve questions of structure, and particularly dynamics at the molecular level.   Weber and collaborators, in papers published from 1980 to the present, demonstrated that most protein made up of subunits can be dissociated by application of hydrostatic pressure, and opened in this way a new method of study of protein aggregates, which is already influencing the approach to problems of both physiology and pathology.  In these studies quite unexpected properties of protein aggregates have been revealed and it is no exaggeration to say that a completely new approach to many related problems in biology and medicine has been suggested by those new observations.  For example, Weber and his collaborators in Urbana, Rio de Janeiro and Göttingen demonstrated the possibility of destroying the infectivity of viruses without affecting their immunogenic capacity and have thus opened the possibility of developing viral vaccines that contain without covalent modification all the antigens present in the original virus.  As a result of his investigations employing fluorescence techniques in conjunction with perturbations by pressure and temperature, Weber presented, in the last few years of his life, a novel way of looking at the folding and association of proteins.  His proposal ran directly contrary to the generally accepted, but notoriously sterile, opinion that the properties of the solvent (water) is the determinant of those phenomena. Instead he proposed that they originate in the very large residual entropy of the protein.

Techniques

  1. Weber, G. Photoelectric method for the measurement of the polarization of the fluorescence of solutions. J. Opt. Soc. Amer. 46, 962-970 (1956).
    Details the one and only no-calibration, absolute method for the measurement of polarization of fluorescence of solutions. Extensions of the method includes: Jameson, D.M., Weber, G., Spencer, R.D. and Mitchell, G. (1978). Fluorescence polarization: Measurements with a photon-counting photometer, Rev. Sci. Instrum. 49(4), 510-514; Chryssomallis, G.S., Drickamer, H.G., and Weber, G. (1978): The measurement of fluorescence polarization at high pressure, J. Appl. Phys. 49(6), 3084-3087; A.A. Paladini and G. Weber (1981): Absolute measurement of polarization of fluorescence at high pressure, Rev. Sci. Inst. 52, 419-426.
  2. Weber, G. and Teale, F.W.J. Determination of the absolute quantum yield of fluorescent solutions. Trans. Faraday Soc. 53, 646-655 (1957).
    Gives what is now the classical method of measurement of absolute quantum yield of fluorescence. Very few values of the many quoted in the original paper have been found to be in error.
  3. Hastings, J.W. and Weber, G. Total quantum flux of isotropic sources. J. Opt. Soc. Am. 53, 1410-1415 (1963).
    An adaptation of method in paper 2 for measurement of quantum yield of chemi- and bioluminescence.
  4. Spencer, R.D. and Weber, G. Measurement of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer. Annals New York Acad. Sci. 158, 361-376 (1969).
  5. Spencer, R.D. and Weber, G. Influence of Brownian rotations and energy transfer upon the measurements of fluorescence lifetime. J. Chem. Phys. 52, 1654-1663 (1970).
  6. Weber, G. Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements. J. Phys. Chem. 85, 949-953 (1981).
  7. Jameson, D.M. and Weber, G. Resolution of the pH dependent heterogeneous fluorescence decay of tryptophan by phase and modulation measurements. J. Phys. Chem. 85, 953-958 (1981).

    Papers 4, 5, 6 and 7 are developments of phase fluorometry. Paper 5 replaces Jablonski's theory and equations, which are incorrect, by the correct ones. Paper 6 gives the theory of resolution of fluorescence decay due to complex independent emissions, a possibility previously limited to pulse fluorometry. Paper 7 applies the theory to a particular case where an independent check of the accuracy can be carried out
    .

  8. Paladini, A.A. and Weber, G. Absolute measurements of fluorescence polarization at high pressures. Rev. Sci. Instrum. 52, 419-427 (1981).
    Describes the extension of the determinations of fluorescence polarization to solutions under pressures of 3 Kbar or less.

Protein-Ligand Interactions

  1. Weber, G. Ligand binding and internal equilibria in proteins. Biochemistry 11, 862 (1972).
  2. Weber, G. Addition of chemical and osmotic free energies through negative interaction of protein bound ligands. Proc. Natl. Acad. Sci. USA 69, 3000-3003 (1972).
  3. Kolb, D.A. and Weber, G. Cooperativity of binding of anilino-naphthalene-sulfonate to serum albumin induced by a second ligand. Biochemistry 14, 4476-4481 (1975).
  4. Weber, G. Energetics of ligand binding to proteins. Adv. Prot. Chem. 29, 1-83 (1975).
  5. Weber, G. Energetic advantage of ion countertransport in chemiosmotic conversion. In Frontiers of Biological Energetics, Academic Press, Vol. 1, pp. 12-18 (1978).
  6. Weber, G. (1992)  Protein Interactions Publishers: Chapman and Hall.

    Papers 1 to 5 describe a general approach to the thermodynamics of multiple ligand binding to proteins, through the simple concept of the "standard free energy couplings" between pairs of bound ligands. This approach is extended to include the covalent reactions in which the protein takes part and can give a rational explanation of the inter conversion of chemical and osmotic energies in metabolism and of the phosphorylation of ADP by ionic gradients. Paper 5 treats explicitly the case of the Na+ K+ ATPase.  Reference 6, a book, is his
    magnum opus on the subject.

  7. Xu, G-J and Weber, G. Dynamics and time-averaged chemical potential of proteins: Importance in oligomer association. Proc. Nat. Acad. Sci USA 79, 5268-5271 (1982).
    The anomalous dissociation of yeast enolase into monomers is explained on the assumption that the chemical potential of the dimer or monomer in equilibrium is not a constant but depends upon the extent of reaction. This is a novel and quite unorthodox concept but may be of great importance in the description of the properties of oligomeric proteins. Careful experimentation will determine whether it has a wider application as we anticipate in this paper.
  8. Weber, G. Asymmetric ligand binding by hemoglobin. Nature 300, 603-607 (1982).
    The relation between asymmetric ligand binding and asymmetric titration curve is developed. The asymmetry observed in the case of hemoglobin is shown to be possible only if the alpha and beta subunit interactions change by different amounts on oxygenation.
  9. Weber, G. Order of free energy couplings between ligand binding and protein subunit association in hemoglobin. Proc. Natl. Acad. Sci. USA 81, 7098-7102 (1984).
  10. Macgregor, R.B. and Weber, G. Estimation of the polarity of the protein interior by optical spectroscopy. Nature 319, 70-73 (1986).
  11. Weber, G. Free energy couplings between ligand binding and subunit association in hemoglobin are of first order. Biochemistry 26, 331-332 (1987).

Development of Fluorescence Probes

  1. Weber, G. (1950) Fluorescence of riboflavin and flavin-adenine dinucleotide. Biochem. J. 47, 114-121.
    First characterization of the fluorescence properties of riboflavin and FAD.
  2. Weber, G. and Laurence, D.J.R. (1954)  Fluorescent indicators of adsorption in aqueous solution and on the solid phase. Biochem. J. 56, xxxi.
    First characterization of the fluorescence properties of ANS.
  3. Weber, G. (1952) Polarization of the fluorescence of macromolecules. II.   Fluorescent conjugates of ovalbumin and bovine serum albumin. Biochem. J. 51, 155-167.
    First synthesis and utilization of dansyl chloride
  4. Weber, G. and Teale, F.W.J. (1957) Ultraviolet fluorescence of aromatic amino acids. Biochem. J. 65, 476-482.
  5. Weber, G. (1960) Fluorescence polarization spectrum and electronic energy transfer in tyrosine, tryptophan and related compounds. Biochem. J. 75, 335-345.
    First characterizationsof the fluorescence properties of tryptophan and tyrosine.
  6. Weber, G. (1957) Intramolecular transfer of electronic energy in dihydrodiphosphopyridine nucleotide. Nature (London) 180, 1409.
    First characterization of the fluorescence properties of NADH.
  7. Knopp, J. and Weber, G. (1967) Fluorescence depolarization measurements in pyrene butyric-bovine serum albumin conjugates. J. Biol. Chem. 242, 1353-1354.
  8. Knopp, A. and Weber, G. (1969) Fluorescence polarization of pyrene-butyric-bovine serum albumin and pyrenebutyric-human macroglobulin conjugates. J. Biol. Chem. 244, 6309-6315.
    Introduction of pyrene as a long lifetime protein probe.
  9. Rosen, C.G. and Weber, G. (1969) Dimer formation from 1-anilino-8-naphthalene sulfonate catalyzed by bovine serum albumin - A new fluorescent molecule with exceptional binding properties. Biochemistry 8, 3915-3920.
  10. Farris, F.J., Weber, G., Chiang, Chian C. and Paul, Iain C. (1978) Preparation, crystalline structure and optical properties of the fluorescent probe, 4-4-bis-1-phenylamino-8-naphthalene sulfonate. J. Amer. Chem. Soc. 100, 4469-4474.
    Introduction and characterization of Bis-ANS.
  11. Hudson, E.N. and Weber, G. (1973) Synthesis and characterization of two fluorescent sulfhydryl reagents. Biochemistry 12, 4154-4161.
    Synthesis and characterization of IAEDANS.
  12. Weber, G. and Farris, F. J. (1979) Synthesis and spectral properties of a hydrophobic fluorescent probe: 2-dimethylamino-6-propionylnaphthalene. Biochemistry 18, 3075-3078.
    Synthesis and characterization of PRODAN - which gave rise to a family of environmentally sensitive probes, also synthesised in the Weber lab, including DANCA and LAURDAN (the popular membrane probe).  ACRYLODAN, synthesized by the Prendergast lab, is also a member of this family of probes.

Study of Interactions of Proteins with Fluorescent Ligands

  1. Weber, G. and Laurence, D.J.R. Fluorescent indicators of adsorption in aqueous solution and on the solid phase. Biochem. J. 56, xxxi (1954).
    Reports the findings that a number of aromatic secondary amines are strongly fluorescent in apolar solvents, but hardly in water, the most spectacular cases being the anilino-naphthalene sulfonates (ANS).
  2. Weber, G. and Daniel, E. Cooperative 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 5, 1900-1907 (1966).
    Describes how polarization measurements may be used to determine the distribution of ligands among the protein molecules that bind them.
  3. Anderson, S.R. and Weber, G. Fluorescence polarization of the complexes of 1-anilino-8-naphthalene sulfonate with bovine serum albumin. Evidence for preferential orientation of the ligand. Biochemistry 8, 371-377 (1969).
  4. Kolb, D.A. and Weber, G. Quantitative demonstration of the reciprocity of ligand effects in the ternary complexes of chicken heart lactate dehydrogenease with NADH and oxalate. Biochemistry 14, 4471 (1975).
    Gives a rigorous quantitative demonstration of the reciprocity of effects among bound ligands. It is shown that in the presence of excess oxalate the free energy of binding of NADH decreases by 1.3 kcal and that of oxalate in the presence of NADH by 1.1 kcal. The figures are equal within experimental errors. Recent work from other laboratories has followed the ideas and technique developed in this study.

U.V. Fluorescence of the Aromatic Amino Acids and Proteins

  1. Weber, G. and Teale, F.W.J. Ultraviolet fluorescence of aromatic amino acids. Biochem. J. 65, 476-482 (1957).
    Paper that first described the fluorescence of the aromatic amino acids.
  2. Weber, G. and Teale, F.W.J. Electronic energy transfer in heme proteins. Faraday Soc. Discussions 27, 134-141 (1959).
    First paper to demonstrate the use of electronic energy transfer in the study of proteins by comparing the fluorescence of heme proteins before and after removal of the heme.
  3. Weber, G. Fluorescence-polarization spectrum and electronic-energy transfer in tyrosine, tryptophan, and related compounds. Biochem. J. 75, 335-345 (1960). 4. Weber, G. Fluorescence-polarization spectrum and electronic-energy transfer in proteins. Biochem. J. 75, 345-352 (1960).

    Papers 3 and 4 - first demonstration of electronic energy transfer among tyrosines, giving the critical distances of transfer from tyrosine to tryptophan and among tyrosine or tryptophan residues.

  4. Lakowicz, J.R. and Weber, G. Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins in the nanosecond time scale. Biochemistry 12, 4171-4179 (1973).
    Describes the technique of using pressures of up to 100 atmospheres of oxygen to quench fluorophores in water. More important, it shows how this can be used to detect, for the first time and to the surprise of many, the existence of fast fluctuations in protein structure on the nanosecond time scale. The relevance of this work is shown in the increasing interest in experimental and theoretical work in protein dynamics.

Effects of hydrostatic pressure upon viruses.

  1. Silva, J.L. and Weber, G. (1988) Pressure-induced dissociation of Brome Mosaic virus. J. Mol. Biol. 199, 149-159.
  2. Silva, J.L., Luan, P., Glaser, M., Voss, E.W. and Weber, G. (1992) Effects of Hydrostatic Pressure on a Membrane-Enveloped Virus: High Immunogenicity of the Pressure-Inactivated Virus. Journal of Virology 66, 2111-2117.
  3. Da Paoian, A.T., Oliveira, A.C., Gaspar, L.P., Silva J.L. and Weber, G. (1993) Reversible pressure dissociation of R17 bacteriophage. The physical individuality of the virus particles. J. Mol. Biol. 231, 999-1008.
  4. Juriekwicz, E., Villas-Boas, M., Silva, J.L., Weber, G.,Hunsmann, G., and Clegg, R.M. (1995) Inactivation of simian immunodeficiency virus by hydrostatic pressure. Proc. Natl. Acad. Sci. USA 92, 6935-6937.
  5. Weber G., Da Poian A. and Silva J.L.(1996) Concentration dependence of the subunit association of oligomers and viruses and the modification of the latter by urea binding. Biophys J. 70, 167-173.

    Papers 1 to 5 provide the first demonstration that viruses are effectively inactivated by the application of hydrostatic pressure in the range of 1-3 kbar. Paper 3 shows that the immunogenicity of vesicular stomatitis viruses is preserved while the infectivity is destroyed under pressure and proves the possibility to prepare by this means effective vaccines that contain all the viral antigens without covalent disruption and in a microscopic form closely resembling that of the intact virus. Paper 4 shows that the simian immunodeficiency virus (SIV) is particularly susceptible to pressure inactivation.

Pressure Effects Upon the Complexes of Small Molecules, Monomeric Proteins and Protein-Ligand Complexes

  1. Weber, G., Tanaka, F., Okamoto, B.Y. and Drickamer, H.G. The effect of pressure on the molecular complex of isoalloxazine and adenine. Proc. Natl. Acad. Sci. USA 71, 1264-1266 (1974).
    Demonstrates that a typical hydrophobic (stacking) complex is stabilized by pressure.
  2. Li, F.M., Hooke, J.W., III, Drickamer, H.G. and Weber, G. Effects of pressure upon the fluorescence of the riboflavin binding protein and its flavin mononucleotide complex. Biochemistry 15, 3205-3211 (1976).
  3. Li, T.M., Hooke, J.W., III, Drickamer, H.G. and Weber, G. Plurality of pressure-denatured forms in lysozyme and chymotrypsinogen. Biochemistry 15, 5517-5580 (1976).
  4. Visser, A.J.W.G., Li, T. M., Drickamer, H.G. and Weber, G. Volume changes in the formation of internal complexes of flavinyltryptophan peptides. Biochemistry 16, 4883-4886 (1977).
  5. Torgerson, P.M., Drickamer, H.G. and Weber, G. Inclusion complexes of poly-b-cyclodextrines. A model for pressure effects upon ligand-protein complexes. Biochemistry 18, 3079-83 (1979).

    Papers 4 and 5 show that the mechanical constraints owing to covalent bonds are important in determining the volume changes upon formation of intramolecular complexes. It is believed that such mechanical constraints are paramount in determining the effects of high pressure upon the monomeric globular proteins.


  6. Torgerson, P.M., Drickamer, H.G. and Weber, G. Effect of hydrostatic pressure upon ethidium bromide association with tRNA. Biochemistry 19, 3957-60 (1980).
  7. Weber, G. and Drickamer, H.G. The effect of high pressure upon proteins and other biomolecules. Quart. Rev. Biophys. 16, 89-112 (1983).

    Papers 1 to 7 form a comprehensive study of pressure effects upon molecular complexes and proteins in solution, carried out in collaboration with Professor H.G. Drickamer at the School of Chemical Sciences, University of Illinois at Urbana. The second paper seems of particular interest in that it demonstrates that "pressure denaturation" of proteins is a complex phenomenon, in which different parts of the protein change conformation over distinctly different pressure ranges.

Thermodynamics of protein condensations

  1. Weber G. (1993) Thermodynamics of the association and the pressure dissociation of oligomeric proteins. J. Phys. Chem. 97, 71108-7115.
  2. Weber, G. (1995) Van't Hoff revisited: Enthalpy of association of protein subunits. J. Phys. Chem. 99, 1052-1059.
  3. Weber, G. (1995) Comment on "van't Hoff revisited: enthalpy of association of protein subunits. J.Phys.Chem. 91,13051.
  4. Weber, G. (1998) Thermodynamic concepts in protein condensation. Comm. Mol. Cell. Biophys. 9. 201-218.

    Papers 1 and 2 present an entirely novel quantitative approach to the thermodynamics of chemical reactions driven by the excess entropy of the products, and apply it to the association of protein subunits of dimers and tetramers. The model adopted is not consistent with the long held ideas on "hydrophobic bonding", in that the entropy increase on subunit association is wholly determined by the intrinsic entropy of the protein and not by the properties of the solvent. Recent measurements on the relaxation of apolar carbons in the protein ubiquitin by NMR (Wand and collaborators) have confirmed that the protein is the seat of the faster motions necessary to assign to it a high residual entropy.

Fluorescence Polarization and Rotational Diffusion

  1. Weber, G. Polarization of the fluorescence of macromolecules. I. Theory and experimental method. Biochem. J. 51, 145-155 (1952). Contains the theory and the method of measurement of the polarization, gives the "polarization addition law", which is the basis for the computation of the polarization of the fluorescence for an arbitrary dipole distribution.
  2. Weber, G. Polarization of the fluorescence of macromolecules. II. Polarization of the fluorescence of labeled protein molecules. Biochem. J. 51, 155-164 (1952). Introduces the Dansyl derivatives as suitable to determine the rotational diffusion of proteins of up to 105 molecular weight, the limit being given by the fluorescence lifetime of Dansyl derivatives.
  3. Knopp, J.A. and Weber, G. Fluorescence polarization of pyrenebutyric bovine serum albumin and pyrenebutyric-human macroglobulin conjugates. J. Biol. Chem. 244, 6309-6315 (1969). Extends the method to molecular weights of l06 (relaxation times of up to 1 ms) by the introduction of a new fluorophore: the pyrene butyroyl residue with a lifetime of 100-150 ns.
  4. Shinitzky, M., Dianoux, A.C., Gitler, C. and Weber, G. Microviscosity and order in the hydrocarbon region of micelles and membranes determined with fluorescent probes. I. Synthetic micelles. Biochemistry 10, 2106-2113 (1971). First paper to describe the use of the fluorescence of small molecules as probes for the viscosity of micelles.
  5. Weber, G. and Mitchell, G.M. "Detection of anisotropic rotations by differential phase fluorometry." In Excited States of Biological Membranes, J.B. Birks (ed.), Wiley, London, pp. 72-76, (1976). The first use of differential phase fluorometry to detect the anisotropic rotations of small molecules.
  6. Weber, G. Theory of differential phase fluorometry: Detection of anisotropic molecular rotations. J. Chem. Phys. 66, 4081-4091 (1977).
    Birth of the theory and experimental proof that it works.
  7. Mantulin, W.W. and Weber, G. Rotational anisotropy and solvent fluorophore bonds: An investigation by differential polarized phase fluorometry. J. Chem. Phys. 66, 4092-4099 (1977).
    A clear demonstration of phenomena previously reported by others that anomalously fast anisotropic rotations may be expected in molecules that do not form strong bonds with the solvent.

Energy Transfer

Besides various papers listed above, which deal with applications, the following two papers were real firsts in this field.

  1. Weber, G. Concentration depolarization of the fluorescence of solutions. Trans. Faraday Soc. 50, 557 (1954).
    Gives the general formulation of depolarization by successive transfers that has been universally adopted afterwards.
  2. Weber, G. and Shinitzky, M. Failure of energy transfer between identical aromatic molecules on excitation at the long wave edge of the absorption spectrum. Proc. Natl. Acad. Sci. USA 65, 823-830 (1970).
    Described the "red edge effect" in energy transfer among identical molecules, amply confirmed by more recent observations.

Effects of Pressure on Oligomeric Proteins

  1. Paladini, A.A. and Weber, G. Pressure-induced reversible dissociation of enolase. Biochemistry 20, 2587-2593 (1981).
  2. King, L. and Weber, G. Conformational drift of lactate dehydrogenase. Biophys.  J. 49, 70-72 (1986).
  3. Weber, G. Phenomenological description of the association of protein subunits subjected to conformational drift. Effects of dilution and of hydrostatic pressure. Biochemistry 25, 3626-3631 (1986).
  4. King, L. and Weber, G. Conformational drift of dissociated lactate dehydrogenase. Biochemistry 25, 3632-3636 (1986).
  5. King, L. and Weber, G. Conformational drift and cryoinactivation of lactate dehydrogenase. Biochemistry 25, 3637-3640 (1986).
  6. Silva, J.L., Miles, E.W. and Weber, G. Pressure dissociation and conformational drift of the ß2 subunit of tryptophan synthase. Biochemistry 25, 578-5786 (1986).
  7. Ruan, K. and Weber, G. (1988) Dissociation of hexokinase. Biochemistry 27, 3295_3301.
  8. Ruan, K. and Weber, G. (1989) Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase. Biochemistry 28, 2144-2153.

    Papers 1 to 8 describe the pressure dissociation of dimers and tetramers and establish the generality of the conformational drift of separated protein subunits.   Papers 5 and 8 propose a new explanation for the inactivation of oligomeric proteins in the cold: A cycle of incipient dissociation, conformational drift of the isolated monomers and reassociation into inactive tetramers that can rearrange themselves into the active form upon warning.

  9. Ruan, K.-C. and Weber, G. (1993) Physical heterogeneity of muscle glycogen phosphorylase revealed by hydrostatic pressure dissociation. Biochemistry 32, 6295-6301.
  10. Erijman, L. and Weber, G. (1991) Oligomeric Protein Associations: Transition from Stochastic to Deterministic Equilibrium. Biochemistry 30, 1595-1599.
  11. Foguel D. and Weber G. (1995) Pressure induced dissociation and denaturation of allophycocyanin at subzero temperatures. J.Biol.Chem. 270, 28759-28766.
  12. Silva, J.L. and Weber, G. (1993) Pressure Stability of proteins. Annu. Rev. Phys. Chem. 44, 89-113.

    Papers 10, 11 and 12 demonstrate the existence of heterogeneity in the free energy of association of tetramers and paper 10 gives a direct demonstration of this heterogeneity employing the transfer of electronic energy among subunits labeled with fluorescent probes. Paper 11 extends the possibilities of use of the dissociating effect of pressure by taking advantage of the decrease of the freezing point of water below 0 centigrade under pressure. Reference 12 reviews the field up to 1993.

Complex Formation of Fluorophores; Detection of Internal Complexes in the Coenzymes

  1. Weber, G. The quenching of fluorescence in liquids by complex formation. Determination of the mean life of the complex. Trans Faraday Soc. 44, 185-189 (1948).
    First paper to demonstrate that fluorescence quenching can take place after formation of molecular complexes of finite duration rather than collisions.
  2. Weber, G. Fluorescence of riboflavin and flavin-adenine dinucleotide. Biochem. J. 47, 114-121 (1950).
    First demonstration of an internal complex in FAD.
  3. Weber, G. Intramolecular transfer of electronic energy in dihydrodiphosphopyridine nucleotide. Nature, London 180, 1409 (1957).
    First demonstration of an internal complex in NADH.

Gregorio Weber - A Fluorescent Lifetime