Gregorio Weber - Research Accomplishments
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.
- 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.
- 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.
- 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
- 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
- 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).
- Weber, G. Resolution of the fluorescence lifetimes in a heterogeneous system by
phase and modulation measurements. J. Phys. Chem. 85, 949-953 (1981).
- 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.
- 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.
- Weber, G. Ligand binding and internal equilibria in proteins. Biochemistry 11, 862 (1972).
- Weber, G. Addition of chemical and osmotic free energies through negative
interaction of protein bound ligands. Proc. Natl. Acad. Sci. USA 69, 3000-3003
- 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).
- Weber, G. Energetics of ligand binding to proteins. Adv. Prot. Chem. 29, 1-83 (1975).
- Weber, G. Energetic advantage of ion countertransport in chemiosmotic conversion. In Frontiers of Biological Energetics, Academic Press, Vol. 1, pp. 12-18 (1978).
- Weber, G. (1992) Protein Interactions Publishers: Chapman and
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.
- 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
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.
- Weber, G. Asymmetric ligand binding by hemoglobin. Nature 300, 603-607
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.
- 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).
- Macgregor, R.B. and Weber, G. Estimation of the polarity of the protein interior by
optical spectroscopy. Nature 319, 70-73 (1986).
- 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
- 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.
- 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.
- Weber, G. (1952) Polarization of the fluorescence of macromolecules. II.
Fluorescent conjugates of ovalbumin and bovine serum albumin. Biochem. J. 51,
First synthesis and utilization of dansyl chloride
- Weber, G. and Teale, F.W.J. (1957) Ultraviolet fluorescence of aromatic amino acids. Biochem. J. 65, 476-482.
- 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.
- Weber, G. (1957) Intramolecular transfer of electronic energy in
dihydrodiphosphopyridine nucleotide. Nature (London) 180, 1409.
First characterization of the fluorescence properties of NADH.
- Knopp, J. and Weber, G. (1967) Fluorescence depolarization measurements in pyrene
butyric-bovine serum albumin conjugates. J. Biol. Chem. 242, 1353-1354.
- Knopp, A. and Weber, G. (1969) Fluorescence polarization of pyrene-butyric-bovine
serum albumin and pyrenebutyric-human macroglobulin conjugates. J. Biol. Chem. 244,
Introduction of pyrene as a long lifetime protein probe.
- 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.
- 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,
Introduction and characterization of Bis-ANS.
- Hudson, E.N. and Weber, G. (1973) Synthesis and characterization of two fluorescent
sulfhydryl reagents. Biochemistry 12, 4154-4161.
Synthesis and characterization of IAEDANS.
- Weber, G. and Farris, F. J. (1979) Synthesis and spectral properties of a
hydrophobic fluorescent probe: 2-dimethylamino-6-propionylnaphthalene. Biochemistry 18,
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
- 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).
- 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.
- 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).
- 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
- 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.
- 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
- 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.
- 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.
- Silva, J.L. and Weber, G. (1988) Pressure-induced dissociation of Brome Mosaic
virus. J. Mol. Biol. 199, 149-159.
- 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.
- 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.
- 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.
- 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
Pressure Effects Upon the Complexes of Small Molecules, Monomeric Proteins and
- 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
- 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).
- 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).
- 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).
- 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.
- Torgerson, P.M., Drickamer, H.G. and Weber, G. Effect of hydrostatic pressure upon
ethidium bromide association with tRNA. Biochemistry 19, 3957-60 (1980).
- 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
- Weber G. (1993) Thermodynamics of the association and the pressure dissociation of
oligomeric proteins. J. Phys. Chem. 97, 71108-7115.
- Weber, G. (1995) Van't Hoff revisited: Enthalpy of association of protein subunits. J.
Phys. Chem. 99, 1052-1059.
- Weber, G. (1995) Comment on "van't Hoff revisited: enthalpy of association of
protein subunits. J.Phys.Chem. 91,13051.
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
Besides various papers listed above, which deal with applications, the following
two papers were real firsts in this field.
- 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.
- 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
- Paladini, A.A. and Weber, G. Pressure-induced reversible dissociation of enolase. Biochemistry
20, 2587-2593 (1981).
- King, L. and Weber, G. Conformational drift of lactate dehydrogenase. Biophys. J. 49, 70-72 (1986).
- 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).
- King, L. and Weber, G. Conformational drift of dissociated lactate dehydrogenase. Biochemistry
25, 3632-3636 (1986).
- King, L. and Weber, G. Conformational drift and cryoinactivation of lactate
dehydrogenase. Biochemistry 25, 3637-3640 (1986).
- 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).
- Ruan, K. and Weber, G. (1988) Dissociation of hexokinase. Biochemistry 27,
- Ruan, K. and Weber, G. (1989) Hysteresis and conformational drift of
pressure-dissociated glyceraldehydephosphate dehydrogenase. Biochemistry 28,
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.
- Ruan, K.-C. and Weber, G. (1993) Physical heterogeneity of muscle glycogen
phosphorylase revealed by hydrostatic pressure dissociation. Biochemistry 32, 6295-6301.
- Erijman, L. and Weber, G. (1991) Oligomeric Protein Associations: Transition from
Stochastic to Deterministic Equilibrium. Biochemistry 30, 1595-1599.
- Foguel D. and Weber G. (1995) Pressure induced dissociation and denaturation of
allophycocyanin at subzero temperatures. J.Biol.Chem. 270, 28759-28766.
- 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
- 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
First paper to demonstrate that fluorescence quenching can take place after
formation of molecular complexes of finite duration rather than collisions.
- Weber, G. Fluorescence of riboflavin and flavin-adenine dinucleotide. Biochem.
J. 47, 114-121 (1950).
First demonstration of an internal complex in FAD.
- 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