Gregorio Weber

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Gregorio Weber (July 4, 1916 – July 18, 1997) was an Argentinian scientist who made significant contributions to the fields of fluorescence spectroscopy and protein chemistry.[1] Weber was elected to the National Academy of Sciences in 1975.

Early life and education[edit]

Gregorio Weber was born in Buenos Aires, Argentina in 1916. He attended the University of Buenos Aires where he received his Doctor of Medicine degree in 1942. While a medical student, from 1939 to 1943, he worked in the Department of Physiology and Biochemistry as a teaching assistant for Bernardo Alberto Houssay who had already achieved renown as a physiologist for his work on the endocrine system and in particular the pituitary gland (Houssay shared the 1947 Nobel Prize for Physiology and Medicine with Carl and Gerty Cori). Weber continued his studies at the University of Cambridge under the guidance of Malcolm Dixon, well-known enzymologist, and, in 1947, earned a Ph.D. in biochemistry. His thesis, titled "Fluorescence of Riboflavin, Diaphorase and Related Substances", marked the beginning of the application of fluorescence spectroscopy to biomolecules.[1] A large portion of Weber’s thesis was devoted to measurements on the quenching of fluorescence of riboflavin and on development of a general theory of quenching by complex formation. This led to his first publication entitled: "The quenching of fluorescence in liquids by complex formation. Determination of the mean life of the complex". This paper was the first to demonstrate that fluorescence quenching can take place after formation of molecular complexes of finite duration rather than collisions. His second publication entitled "Fluorescence of riboflavin and flavin adenine dinucleotide", was the first demonstration of an internal complex in FAD. Years later he was to follow up this work with the first demonstration that NADH also formed an internal complex and with more complete characterizations of the excited state properties of FAD and NADH. From 1948 to 1952 Weber carried out independent investigations at the Sir William Dunn Institute of Biochemistry at Cambridge, supported by a British Beit Memorial Fellowship. At that time he began to delve more deeply into the theory of fluorescence polarization and also began to develop methods which would allow him to study proteins which did not contain an intrinsic fluorophore such as FAD or NADH (the fluorescence of the aromatic amino acids had not yet been discovered). To this end, he invested considerable time and effort in synthesizing a fluorescent probe which could be covalently attached to proteins and which possessed absorption and emission characteristics appropriate for the instrumentation available in post-war England. The result of two years of effort was the still popular probe dimethylaminonaphthalene sulfonyl chloride or dansyl chloride. With this tool in hand and with new instrumentation he began to investigate several protein systems, publishing his theory and experimental results in two classic papers published in 1952, namely, Polarization of the fluorescence of macromolecules. I. Theory and experimental method and Polarization of the fluorescence of macromolecules. II. Fluorescent conjugates of ovalbumin and bovine serum albumin. The theory paper (which contains an acknowledgement to F. Perrin for his suggestions) includes an extension of Perrin’s theory of depolarization due to rotation of ellipsoidal molecules. Specifically, Weber showed that Perrin’s complex equations, which required a knowledge of the orientation of the fluorophore’s absorption and emission oscillators with respect to the axis of rotation of the ellipsoid, could be considerably simplified if the fluorophores carrying the oscillators were assumed to be randomly oriented on the macromolecule. This paper also contained a formulation of the law of additivity of polarizations. Weber stayed at Cambridge as an independent researcher until 1953 when Hans Krebs recruited him for the new Biochemistry Department at Sheffield University.

The Sheffield years[edit]

During his years at Sheffield, Weber continued to lay the foundations of modern fluorescence spectroscopy developing both fluorescence theory and instrumentation. His pioneering contributions during these early years included his report with Laurence of aromatic secondary amines, which were strongly fluorescent in apolar solvents but very weakly fluorescent in water, the most spectacular case being the anilino naphthalene sulfonates (ANS). More than 50 years after that first report, ANS is still being used in protein studies, quite often as an indicator of the “molten globule state.” During those early years at Sheffield, Weber and his postdoctoral fellow, F.W. John Teale, began their studies on intrinsic protein fluorescence. At that time, compounds resembling the aromatic amino acids had been shown to possess appreciable fluorescence in the near ultraviolet but the fluorescence of the aromatic amino acids themselves had not yet been unequivocally characterized (although Debye and Edwards had made observations on the phosphorescence of the aromatic amino acids and the position of these phosphorescence bands indicated to McClure the probable existence of fluorescence bands in the near ultraviolet). In 1953, Weber hypothesized that emission bands for tyrosine and tryptophan should exist with maxima in the region 3000 - 4000 Å. At about the same time Weber and Teale were carrying out their studies, Shore and Pardee adapted a Beckman DU spectrophotometer to view the ultraviolet fluorescence of tyrosine, tryptophan and a number of proteins through a filter that passed wavelengths greater than about 300 nm. Shore and Pardee could not record emission spectra with their apparatus, however, and the excitation spectra obtained were very approximate. In 1957, Weber and Teale published the first emission spectra of the aromatic amino acids, and the first accurate excitation spectra (figure 7 from this paper has been reproduced many times). In the late 1950s and early 1960s, Weber and Teale published a series of important papers and communications on intrinsic protein fluorescence and the determination of absolute quantum yields. The quantum yield Weber and Teale reported for tryptophan, 0.20, was later found to be somewhat higher than the currently accepted value near 0.14. At the time Weber and Teale carried out their experiments, however, the large temperature effect on tryptophan’s lifetime and quantum yield was not appreciated and their work, reported as done at “room temperature”, was in fact carried out in the winter in a Quonset hut without heating, which caused a marked increase in their tryptophan quantum yield relative to that expected for 25°C. In 1960, Weber published the excitation polarization spectra of the aromatic amino acids and numerous proteins and also gave the first demonstration of electronic energy transfer among tyrosines and tryptophans and the critical transfer distances from tyrosine to tryptophan and among tyrosine or tryptophan residues. In 1959, Weber and Teale also demonstrated the first use of electronic energy transfer in the study of hemeproteins by comparing the fluorescence of hemoglobin and horseradish peroxidase before and after removal of the heme. During the four decades since the first description of protein fluorescence, thousands of papers have been written on the fluorescence of tryptophan, tyrosine or phenylalanine or some aspect of intrinsic protein fluorescence. The study of intrinsic protein fluorescence has, in fact, become one of the most important techniques used in protein research and has been of great importance in establishing the dynamic nature of proteins. This potential was certainly not lost on Weber who presented a classic paper at the “Light and Life” conference held in 1960 and, in a true understatement, summarized his presentation by saying “There are many ways in which the properties of the excited state can be utilized to study points of ignorance of the structure and function of proteins”. In fact, in an earlier communication (presented at the annual meeting of the British Biochemical Society on April 3, 1959) Weber estimated that the excited state lifetime of tryptophan in proteins was on the order of 4 ns and commented “These values are too short to permit measurements of fluorescence polarization to be of value in the determination of the rotational relaxation times of proteins in solution, but can give useful information on local conditions about the tryptophan or tyrosine residues.” Now that present day methods of site-directed mutagenesis permit the facile removal and/or addition of tryptophan residues to allow the creation of novel single-tryptophan containing proteins, Weber’s vision of the utility of intrinsic protein fluorescence is being fully realized.

University of Illinois[edit]

In the early 1960s, I.C. “Gunny” Gunsalus, then the head of the Biochemistry Division of the Department of Chemistry at the University of Illinois at Urbana-Champaign, recruited Weber. Gunny related the story that while he was convincing his colleagues that Gregorio Weber was an exceptional scientist, someone commented that Weber didn’t have as many publications as one might expect from a senior professor. Gunny explained that while this was true, Weber’s ratio of outstanding papers to total papers was unity and that this ratio - known thereafter as the Weber ratio - was certainly the more important consideration. Gregorio Weber joined the University of Illinois in 1962 and built a research program that continued actively until his death from leukemia on July 17, 1997. During the early years in Urbana, Weber continued to develop novel fluorescence instrumentation and probes and extended his studies of protein systems.

Scientific contributions[edit]

Gregorio Weber is acknowledged to be the person responsible for many of the more important theoretical and experimental developments in modern fluorescence spectroscopy. In particular, Weber pioneered the application of fluorescence spectroscopy to the biological sciences. His list of achievements includes: the synthesis and use of dansyl chloride as a probe of protein hydrodynamics; the extension of Perrin’s theory of fluorescence polarization to fluorophores associated with random orientations with ellipsoids of revolution and to mixtures of fluorophores; the first spectral resolution of the fluorescence of the aromatic amino acids and of intrinsic fluorescence of proteins; the first demonstration that both FAD and NADH make internal complexes; the first report on aromatic secondary amines, which are strongly fluorescent in apolar solvents, but hardly in water, the most spectacular case being the anilino naphthalene sulfonates (ANS); the first description of the use of the fluorescence of small molecules as probes for the viscosity of micelles, with implications for membrane systems; a general formulation of depolarization by energy transfer; the discovery of the “red-edge” effect in homo-energy transfer; the development of modern cross-correlation phase fluorometry; the development of the excitation-emission matrix method for resolving contributions from multiple fluorophores; the synthesis of several novel fluorophores, including pyrenebutyric acid, IAEDANS, bis-ANS, PRODAN and LAURDAN, designed to probe dynamic aspects of biomolecules. In addition to these seminal contributions, Gregorio Weber also trained and inspired generations of spectroscopists and biophysicists who went on to make important contributions to their fields, including both basic research as well as the commercialization of fluorescence methodologies and their extension into the clinical and biomedical disciplines.

Gregorio Weber’s original and lifelong motivation was to use fluorescence methods to probe the nature of proteins and in addition to his contributions to the fluorescence field, he was one of the true pioneers of protein dynamics. A study of his papers from the 1960s demonstrates that even then he regarded proteins as highly dynamic molecules. He rejected the view, common at that time after the appearance of the first x-ray structures, that proteins had a unique and rigid conformation. In an important innovation, he introduced the use of molecular oxygen to quench fluorescence in aqueous solutions, which led to the detection, for the first time and to the surprise of many, of the existence of fast fluctuations in protein structures on the nanosecond time scale. The impact of this work was shown by the increasing interest in experimental and theoretical work in protein dynamics, which followed. Weber’s early description of proteins in solution as “kicking and screaming stochastic molecules” has, in recent years, been fully verified both from theoretical and experimental studies. These contributions were recognized by the American Chemical Society in 1986, which named Weber as the first recipient of Repligen Award for the Chemistry of Biological Processes. In the 1970s, initially in collaboration with H.G. Drickamer, Weber combined fluorescence and hydrostatic pressure methods to the study of molecular complexes and proteins. The initial system he thought to study was the complex formed by isoalloxazine and adenine, one of his original research interests. These observations confirmed the applicability of fluorescence and high-pressure techniques to problems of structure, and particularly dynamics, at the molecular level. Weber and collaborators demonstrated that most proteins made up of subunits can be dissociated by the application of hydrostatic pressure, and opened, in this way, a new method to study protein-protein interactions. In these studies, quite unexpected properties of protein aggregates were revealed and a new approach to problems in biology and medicine was opened by these observations. For example, Weber and his collaborators demonstrated the possibility of destroying the infectivity of viruses, without affecting their immunogenic capacity, by subjecting them to hydrostatic pressure, and thus opened the possibility of developing viral vaccines that contain, without covalent modification, all the antigens present in the original virus.


Gregorio Weber’s scientific achievements were recognized by many honors and awards. These include election to the US National Academy of Sciences, election to the American Academy of Arts and Sciences, election as a corresponding member to the National Academy of Exact Sciences of Argentina, the first National Lecturer of the Biophysical Society, the Rumford Premium of the American Academy of Arts and Sciences, the ISCO Award for Excellence in Biochemical Instrumentation, the first Repligen Award for the Chemistry of Biological Processes (awarded by the American Chemical Society) and the first International Jablonski Award for Fluorescence Spectroscopy. It is worth noting that the Rumford Premium is one of the oldest scientific awards given in the United States. It was created by a bequest to the Academy from Benjamin Thompson, Count Rumford, in 1796 - previously awardees include J. Willard Gibbs, A.A. Michelson, Thomas Edison, R.W. Wood, Percy Bridgman, Irving Langmuir, Enrico Fermi, S. Chandrasekhar, Hans Bethe, Lars Onsanger and other highly original thinkers. The Rumford award committee recommended that the 1979 award be given to two physicists, Robert L. Mills and Chen Ning Yang, for their joint work on the theory of gauge invariance of the electromagnetic field, and to Gregorio Weber, “Acknowledged to be the person responsible for modern developments in the theory and application of fluorescent techniques to chemistry and biochemistry”.

To honor Gregorio Weber, approximately every three years the "Weber Symposia" are held. These international symppsia are now held on the island of Kauai, in Hawaii. The last symposium was held during June 15-20, 2014 and had participants from 18 countries.


  1. ^ a b "Biophysical Journal, Volume 75, July 1998, pages 419-421"

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