[1]
REFLECTIONS
3
[1] R e f l e c t i o n s
By FREDERIC M. RICHARDS Avidin was recognized as a biological factor in egg white in the late 1920s during the discovery and isolation of the vitamin biotin.l The latter compound joined the other known water-soluble vitamins, which received interest both because of the related deficiency diseases and because of the emerging identification of vitamins as cofactors in various metabolic processes. Avidin activity was found in various avian eggs and egg jelly of invertebrates but could not be found in many other tissues or organisms. Its function in the egg was presumed to be that of an antibiotic along with several other proteins of similar function but very different mechanisms, lysozyme, for example. With no general biological function, and no apparent clinical use as an antibiotic, avidin was essentially ignored for about two decades. In the late 1950s the laboratories of Wakil and Lynen both reported the discovery of covalently bound biotin with a coenzyme function. 2 Avidin at once took on the role of a tool in the investigation of this new class of enzymes. The use of avidin in the study of the biotin enzymes appeared suitable for that purpose but of limited general application. Shortly afterward Green began studies of the detailed protein chemistry of avidin, focusing on its general structure and activity rather than its use as a reagent. 3 Of particular interest since the earliest days has been the remarkable strength of the interaction between avidin and biotin. The binding is characterized by a dissociation constant of the order of 10 J5 M. This value corresponds to a free energy of association of about 21 kcal/mol, a staggeringly large value for the noncovalent interaction of a protein with a molecule as small as biotin. In general such binding is found only in systems involving liganded metal ions either as partial covalent bonds or chelates. No metal ions are involved in the avidin-biotin binding. Of special importance is the very slow off-rate that accompanies such a tight association. i F. K6gl and B. T6nnis, Z. Physiol. Chem. 242, 43 (1936). F. Lynen, J. Knappe, E. Lorch, G. Jutting, and E. Ringelmann, Angew. Chem. 71, 481 (1959); S. J. Wakil and D. M. Gibson, Biochim. Biophys. Acta 41, 122 (1960). 3 N. M. Green, Adv. Protein Chem. 29, 85 (1975).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
INTRODUCTION
[1]
While these chemical studies were proceeding, Chaiet and colleagues 4 reported avidinlike activity from various species of Streptomyces, one more, apparently, in the series of antibiotic materials produced by these bacteria. While similar to egg avidin in many ways, streptavidin has a much more acidic isoelectric point. Again, with no clinical application this substance appeared to be a curiosity, and interest was slow in developing. The future importance of this form of avidin was certainly not clear at the beginning. In the early to mid-1970s a series of events occurred which appear to have changed the perception of the avidin-biotin system. John Edsall, Chris Anfinsen, and I, as editors of Advances in Protein Chemistry, asked Michael Green to write a review article on avidin. The article contained all of the detailed protein chemical information then available on avidin. The evidence for a tetramer with the four biotin-binding sites arranged in two clusters was presented. The clever cross-linking experiments with bisbiotin samples of varying chain length defined the deep binding pocket and recessed surface area in which it lies. The binding constants for a large number of biotin derivatives were collected. The review 3 was published in 1975 and served as a background for subsequent applications. The major published studies of Green on the properties of the avidinbiotin system suggested to Heitzmann and myself the possibility of developing a general tool for labeling membranes, 5 while others were applying the system for various other purposes. 6 The convenient single carboxyl group on biotin and the relatively low reactivity of the fused ring system made the preparation of reagents for the chemical modification of proteins straightforward. Because of the enormous binding constant we thought of the whole system as sort of a chemist's version of an antigen-antibody labeling procedure, to which, in fact, it is a complement. We did note that the very slow off-rate would allow sequential labeling of different reactive groups using different heavy metal tags, but we did not envision the use of this system for separations. Because of the close association of our laboratories, Wallace and Engelman used this labeling procedure, shortly after its development, to study general protein distribution and redistribution during the phase transition in Acholeplasma membranes. 7
4 L. Chaiet, T. W. Miller, F. Tausig, and F. J. Wolf, Antimicrob. Agents Chemother. 3, 28 (1963); L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). 5 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). 6 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Lett. 68, 240 (1976); E. A. Bayer and M. Wilchek, Trends Biochem. Sci. 3, N257 (1978); K. Hofmann, F. M. Finn, and Y. Kiso, J. Am. Chem. Soc. 100, 3585 (1978). 7 B. A. Wallace, F. M. Richards, and D. M. Engelman, J. Mol. Biol. 107, 255 (1976).
[2]
INTRODUCTION
TO AVIDIN--BIOTIN
TECHNOLOGY
5
Completely independently, Tom Broker and Norman Davidson at the California Institute of Technology had the same general ideas for using the avidin-biotin system, and they developed procedures for gene mapping by labeling RNA molecules, especially tRNA, and then using them as hybridization probes to locate genes in double-stranded DNA. 8 After hearing about this work at meetings, we did have some casual discussions with David Ward in Human Genetics at Yale, University. 9 It was clear that the biotin-avidin system as a general cytochemical labeling procedure had arrived. 10 I doubt that any of us at the time imagined the explosion in applications that would occur over the 1980s ~1 and that is documented in this volume. With the basic underpinnings for the technology now in hand, as well as cloned genes for the avidins ~2 and crystal structures of the proteins, 13 the future is bright indeed. 8 j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975); T. R. Broker, L. M. Angerer, P. Yen, N. D. Hershey, and N. Davidson, Nucleic Acids Res. 5, 363 (1978). 9 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). to E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980); M. Wilchek and E. A. Bayer, lmrnunol. Today 5, 39 (1984). 11 M. Wilchek and E. A. Bayer, Anal. Biochem. 171, 1 (1988). t2 M. L. Gope, R. A. Keinanen, P. A. Kristo, O. M. Conneely, W. G. Beattie, T. ZaruckiSchulz, B. M. O'Malley, and M. S. Kulomaa, Nucleic Acids. Res. 15, 3595 (1987); G. Chandra and J. G. Gray, this volume [7]; C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). ~3 E. Pinn, A. P/ihler, W. Saenger, G. Petsko, and N. M. Green, Eur. J Biochem. 123, 545 (1982); A. P~ihler, W. A. Hendrickson, M. A. G. Kolks, C. E. Argarana, and C. R. Cantor, J. Biol. Chem. 262, 13933 (1987).
[2] I n t r o d u c t i o n to A v i d i n - B i o t i n T e c h n o l o g y
By MEIR WILCHEK and EDWARD A. BAYER The avidin-biotin system has many uses in both research and technology. This general introductory chapter presents an overview of the principles and advantages of this system, and illustrates the numerous applications of avidin-biotin technology. It also explains in general and explicit terms the basic idea behind avidin-biotin technology. METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
REFLECTIONS
3
[1] R e f l e c t i o n s
By FREDERIC M. RICHARDS Avidin was recognized as a biological factor in egg white in the late 1920s during the discovery and isolation of the vitamin biotin.l The latter compound joined the other known water-soluble vitamins, which received interest both because of the related deficiency diseases and because of the emerging identification of vitamins as cofactors in various metabolic processes. Avidin activity was found in various avian eggs and egg jelly of invertebrates but could not be found in many other tissues or organisms. Its function in the egg was presumed to be that of an antibiotic along with several other proteins of similar function but very different mechanisms, lysozyme, for example. With no general biological function, and no apparent clinical use as an antibiotic, avidin was essentially ignored for about two decades. In the late 1950s the laboratories of Wakil and Lynen both reported the discovery of covalently bound biotin with a coenzyme function. 2 Avidin at once took on the role of a tool in the investigation of this new class of enzymes. The use of avidin in the study of the biotin enzymes appeared suitable for that purpose but of limited general application. Shortly afterward Green began studies of the detailed protein chemistry of avidin, focusing on its general structure and activity rather than its use as a reagent. 3 Of particular interest since the earliest days has been the remarkable strength of the interaction between avidin and biotin. The binding is characterized by a dissociation constant of the order of 10 J5 M. This value corresponds to a free energy of association of about 21 kcal/mol, a staggeringly large value for the noncovalent interaction of a protein with a molecule as small as biotin. In general such binding is found only in systems involving liganded metal ions either as partial covalent bonds or chelates. No metal ions are involved in the avidin-biotin binding. Of special importance is the very slow off-rate that accompanies such a tight association. i F. K6gl and B. T6nnis, Z. Physiol. Chem. 242, 43 (1936). F. Lynen, J. Knappe, E. Lorch, G. Jutting, and E. Ringelmann, Angew. Chem. 71, 481 (1959); S. J. Wakil and D. M. Gibson, Biochim. Biophys. Acta 41, 122 (1960). 3 N. M. Green, Adv. Protein Chem. 29, 85 (1975).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
INTRODUCTION
[1]
While these chemical studies were proceeding, Chaiet and colleagues 4 reported avidinlike activity from various species of Streptomyces, one more, apparently, in the series of antibiotic materials produced by these bacteria. While similar to egg avidin in many ways, streptavidin has a much more acidic isoelectric point. Again, with no clinical application this substance appeared to be a curiosity, and interest was slow in developing. The future importance of this form of avidin was certainly not clear at the beginning. In the early to mid-1970s a series of events occurred which appear to have changed the perception of the avidin-biotin system. John Edsall, Chris Anfinsen, and I, as editors of Advances in Protein Chemistry, asked Michael Green to write a review article on avidin. The article contained all of the detailed protein chemical information then available on avidin. The evidence for a tetramer with the four biotin-binding sites arranged in two clusters was presented. The clever cross-linking experiments with bisbiotin samples of varying chain length defined the deep binding pocket and recessed surface area in which it lies. The binding constants for a large number of biotin derivatives were collected. The review 3 was published in 1975 and served as a background for subsequent applications. The major published studies of Green on the properties of the avidinbiotin system suggested to Heitzmann and myself the possibility of developing a general tool for labeling membranes, 5 while others were applying the system for various other purposes. 6 The convenient single carboxyl group on biotin and the relatively low reactivity of the fused ring system made the preparation of reagents for the chemical modification of proteins straightforward. Because of the enormous binding constant we thought of the whole system as sort of a chemist's version of an antigen-antibody labeling procedure, to which, in fact, it is a complement. We did note that the very slow off-rate would allow sequential labeling of different reactive groups using different heavy metal tags, but we did not envision the use of this system for separations. Because of the close association of our laboratories, Wallace and Engelman used this labeling procedure, shortly after its development, to study general protein distribution and redistribution during the phase transition in Acholeplasma membranes. 7
4 L. Chaiet, T. W. Miller, F. Tausig, and F. J. Wolf, Antimicrob. Agents Chemother. 3, 28 (1963); L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). 5 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). 6 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Lett. 68, 240 (1976); E. A. Bayer and M. Wilchek, Trends Biochem. Sci. 3, N257 (1978); K. Hofmann, F. M. Finn, and Y. Kiso, J. Am. Chem. Soc. 100, 3585 (1978). 7 B. A. Wallace, F. M. Richards, and D. M. Engelman, J. Mol. Biol. 107, 255 (1976).
[2]
INTRODUCTION
TO AVIDIN--BIOTIN
TECHNOLOGY
5
Completely independently, Tom Broker and Norman Davidson at the California Institute of Technology had the same general ideas for using the avidin-biotin system, and they developed procedures for gene mapping by labeling RNA molecules, especially tRNA, and then using them as hybridization probes to locate genes in double-stranded DNA. 8 After hearing about this work at meetings, we did have some casual discussions with David Ward in Human Genetics at Yale, University. 9 It was clear that the biotin-avidin system as a general cytochemical labeling procedure had arrived. 10 I doubt that any of us at the time imagined the explosion in applications that would occur over the 1980s ~1 and that is documented in this volume. With the basic underpinnings for the technology now in hand, as well as cloned genes for the avidins ~2 and crystal structures of the proteins, 13 the future is bright indeed. 8 j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975); T. R. Broker, L. M. Angerer, P. Yen, N. D. Hershey, and N. Davidson, Nucleic Acids Res. 5, 363 (1978). 9 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). to E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980); M. Wilchek and E. A. Bayer, lmrnunol. Today 5, 39 (1984). 11 M. Wilchek and E. A. Bayer, Anal. Biochem. 171, 1 (1988). t2 M. L. Gope, R. A. Keinanen, P. A. Kristo, O. M. Conneely, W. G. Beattie, T. ZaruckiSchulz, B. M. O'Malley, and M. S. Kulomaa, Nucleic Acids. Res. 15, 3595 (1987); G. Chandra and J. G. Gray, this volume [7]; C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). ~3 E. Pinn, A. P/ihler, W. Saenger, G. Petsko, and N. M. Green, Eur. J Biochem. 123, 545 (1982); A. P~ihler, W. A. Hendrickson, M. A. G. Kolks, C. E. Argarana, and C. R. Cantor, J. Biol. Chem. 262, 13933 (1987).
[2] I n t r o d u c t i o n to A v i d i n - B i o t i n T e c h n o l o g y
By MEIR WILCHEK and EDWARD A. BAYER The avidin-biotin system has many uses in both research and technology. This general introductory chapter presents an overview of the principles and advantages of this system, and illustrates the numerous applications of avidin-biotin technology. It also explains in general and explicit terms the basic idea behind avidin-biotin technology. METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
