Photoaffinity Labeling of Proteins and More Complex Receptors

  • Barry S. Cooperman


In the past several years affinity labeling has emerged as a powerful method for studying ligand-receptor interactions. The logic of an affinity labeling experiment (Singer, 1967) is described in Eq. (1)–(3). A reactive group, X (electrophilic or photolabile), is attached to the natural ligand, L. If the resulting modified ligand ĹX retains high affinity for the native ligand receptor site and forms a non-covalent complex, R.ĹX, [Eq. (1)], then, at low ĹX concentration, covalent attachment via the reaction in Eq. (2), a first order process, will proceed at a much faster rate than attachment via the reaction in Eq. (3), a second order process, and specific attachment to the ligand site will be achieved.


Label Pattern Noncovalent Complex Diazo Compound Photoaffinity Label Covalent Bond Formation 
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  1. Anderson, E., Nakashima, Y., and Konigsberg, W., 1975, Photo-induced cross-linkage of gene-5 protein and bacteriophage fd DNA, Nucleic Acids Res. 2: 361–371.PubMedCrossRefGoogle Scholar
  2. Antonoff, R., and Ferguson, Jr., J.J., 1974, Photoaffinity labeling with cyclic nucleotides, J. Biol. Chem. 249: 3319–3321.PubMedGoogle Scholar
  3. Baron, W.J., DeCamp, M.R., Henrick, M.A., Jones, Jr., M., Levin, R.H., and Sohn, M.B., 1973, Carbenes from diazo compounds in “Carbenes” (M. Jones, Jr., and R.A. Moss, eds.), Vol. I, pp. 1–151, WileyInterscience, New York.Google Scholar
  4. Bispink, L., and Matthaei, H., 1973, Photoaffinity labeling of 23S RNA in E.coli ribosomes with poly (U)-coded ethyl-2-diazomalonyl phenylalanyl-tRNA, FEBS Lett. 37: 291–294.PubMedCrossRefGoogle Scholar
  5. Brandt, J., Fredriksson, M., and Anderson, L.-O., 1974, Coupling of dyes to biopolymers by sensitized photooxidation affinity labeling of a binding site in bovine serum albumin, Biochemistry 13: 4758–4764.PubMedCrossRefGoogle Scholar
  6. Breslow, R., Baldwin, S., Flechtner, T., Kalicky, P., Liu, S., and Washburn, W., 1973, Remote oxidation of steroids by photolysis of attached benzophenone groups, J. Amer. Chem. Soc. 95: 3251–3262.CrossRefGoogle Scholar
  7. Breslow, R., Feiring, A., and Herman, F., 1974, Intramolecular insertion reactions of phosphoryl nitrenes, J. Amer. Chem. Soc. 96: 5937–5939.CrossRefGoogle Scholar
  8. Bridges, A.J., and Knowles, J.R., 1974, An examination of the utility of photogenerated reagents by using a-chymotrypsin, Biochem. J. 143: 663–668.PubMedGoogle Scholar
  9. Budker, V.G., Knorre, O.G., Kravchenko, U.Va., Lavrik, O. II, Nevinsky, G.A., and Teplova, N.M., 1974, Photoaffinity reagents for modification of amino acyl-tRNA synthetases, FEBS Lett. 49: 159–162.PubMedCrossRefGoogle Scholar
  10. Cannon, I.E., Woodward, D.K., Woehler, M.E., and Lovins, R.E., 1974, Affinity labeling of isoelectrofocused fractions from a DNP antibody preparation with the photoactive labels 2,4-dinitrophenyl-1-azide and 2,4-dinitrophenyl-c-aminocaproyldiazoketone, Immunology 26: 1183–1194.PubMedGoogle Scholar
  11. Gloss, G.L., 1975, Application of physical methods to the study of carbene reactions in solution, in “Carbenes”, (R.A. Moss, and M. Jones, Jr., eds), Vol. II, pp. 159–183, Wiley-Interscience, New York.Google Scholar
  12. Cooperman, B.S., and Brunswick, D.J., 1973, On the photoaffinity labeling of rabbit muscle phosphofructokinase with O2’-(ethyl2-diazomalonyl)adenosine 3’:5’-cyclic monophosphate, Biochemistry 12: 4079–4084.PubMedCrossRefGoogle Scholar
  13. Cooperman, B.S., Jaynes, Jr., E.N., Brunswick, D.J., and Luddy, M.A., 1975, On the photoincorporation of puromycin and N(ethyl-2-diazomalonyl)-puromycin into E. coli ribosomes, Proc. Nat. Acad. Sci. USA 72 (in press).Google Scholar
  14. Creed, D., 1974, Photochemical probes for biological interactions, Photochem. Photobiol. 19: 459–462.PubMedCrossRefGoogle Scholar
  15. Cysyk, R., and Prusoff, W.H., 1972, Alteration of ultraviolet sensitivity of thymidine kinase by allosteric regulators, normal substrates, and a photoaffinity label, 5-iodo-2’deoxyuridine, a metabolic analog of thymidine, J. BioZ. Chem. 247: 2522–2532.Google Scholar
  16. Escher, E., and Schwyzer, R., 1974, p-Nitrophenylalanine-p-azidophenylalanine, m-azidophenylalanine, and o-nitro-p-azidophenylalanine as photoaffinity labels, FEBS Lett. 46: 347–350.PubMedCrossRefGoogle Scholar
  17. Fiser, I., Margaritella, P., and Kuechler, E., 1975a, Photoaffinity reaction between polyuridylic acid and protein S1 on the E. coli ribosome, FEBS Lett. 52: 281–283.PubMedCrossRefGoogle Scholar
  18. Fiser, I., Scheit, K.H., Stöffler, G., and Kuechler, E., 1974, Identification of protein S1 at the messenger RNA binding site of E.coli ribosome, Biochem. Biophys. Res. Commun. 60: 1112–1118.PubMedCrossRefGoogle Scholar
  19. Fiser, I., Scheit, K.H., Stöffler, G., and Kuechler, E., 1975b, Proteins at the mRNA binding site of the E.coli ribosome, FEBS Lett.,In Press.Google Scholar
  20. Fisher, C.E., and Press, E.M., 1974, Affinity labeling of the binding site of rabbit antibody-evidence for the involvement of the hypervariable regions of the heavy chain, Biochem. J. 139: 135–149.PubMedGoogle Scholar
  21. Fleet, G.W.J., Knowles, J.R., and Porter, R.R., 1972, The antibody site-labeling of a specific antibody against the photo-precursor of an aryl nitrene, Biochem. J. 128: 499–508.PubMedGoogle Scholar
  22. Frischauf, A.M. and Scheit, K.H., 1973, Affinity labeling of E.coZi RNA polymerase with substrate and template analogs, Biochem. Biophys. Res. Commun. 53: 1227–1233.PubMedCrossRefGoogle Scholar
  23. Galardy, R.E., Craig, L.C., Jamieson, J.D., and Printz, M.P., 1974, Photoaffinity labeling of peptide hormone binding sites, J. BioZ. Chem. 249: 3510–3518.Google Scholar
  24. Glover, G.I., Mariano, P.S., Wilkinson, T.J. Hildreth, R.A., and Lowe, T.W., 1974, Photofragmentation and photoaffinity labeling of phenacyl and naphthacyl a-chymotrpsins, Arch. Biochem. Biophys. 162: 73–83.PubMedCrossRefGoogle Scholar
  25. Greenwell, P., Jewett, S.L., and Stark, G.R., 1973, Aspartate transcarbamylase from E.coZi. The use of pyridoxal 5’phosphate as a probe in the active site, J. BioZ. Chem. 248: 5994–6001.Google Scholar
  26. Guthrow, C.E., Rasmussen, H., Brunswick, D.J., and Cooperman, B.S., 1973, Specific photoaffinity labeling of the adenosine 3’:5’-cyclic monophosphate receptor in intact ghosts from human erythrocytes, Proc. Nat. Acad. Sci. USA, 70: 3344–3346.PubMedCrossRefGoogle Scholar
  27. Haley, B.E., and Hoffman, J.F., 1974, Interactions of a photo-affinity ATP analog with cation-stimulated adenosine triphosphatases of human red cell membranes, Proc. Nat. Acad. Sci. USA 71: 3367–3371.PubMedCrossRefGoogle Scholar
  28. Hanstein, W.G., and Hatefi, Y., 1974, Characterization and localization of metochondrial uncoupler binding sites with an un-coupler capable of photoaffinity labeling, J. Biol. Chem. 249: 1356–1362.PubMedGoogle Scholar
  29. Hexter, C.S., and Westheimer, F.H., 1971a, Intermolecular reaction during photolysis of diazoacetyl a-chymotrypsin, J. Biol. Chem. 246: 3928–3933.PubMedGoogle Scholar
  30. Hexter, C.S., and Westheimer, F.H., 1971b, S-carboxymethylcysteine from the photolysis of diazoacyl trypsin and chymotrypsin, J. Biol. Chem. 246: 3924–3928.Google Scholar
  31. Hew, C-L., Lifter, J., Yoshioka, M., Richards, F.F., and Konigsberg, W.H., 1973, Affinity labeled peptides obtained from the combining region of protein 460. Light chain labeling patterns using dinitrophenyl based photoaffinity labels, Biochemistry 12: 4685–4689.PubMedCrossRefGoogle Scholar
  32. Nixon, S.C., White, Jr., W.E., and Yielding, K.L., 1975, Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labeling, J. Mol. Biol. 92: 319–329.CrossRefGoogle Scholar
  33. Hixson, S.S., and Hixson, S.H., 1972, The photochemistry of s-methyl diazothioacetate, J. Org. Chem. 37: 1279–1280.CrossRefGoogle Scholar
  34. Hsiung, N., and Cantor, C.R., 1974, A new simpler photoaffinity analogue of peptidyl tRNA, Nucleic Acids Res. 1: 1753–1762.PubMedCrossRefGoogle Scholar
  35. Hsiung, N., Reines, S.A., and Cantor, C.R., 1974, Investigation of the ribosomal peptidyl transferase center using a photoaffinity label, J. Mol. Biol. 88: 841–855.PubMedCrossRefGoogle Scholar
  36. Hucho, F., Markau, U., and Sund, H., 1973, Studies of glutamate dehydrogenase characterization of histidine residues involved in the activity and association photoactivated labelling with pyridoxal 5’-phosphate, Eur. J. Biochem. 32: 69–75.PubMedCrossRefGoogle Scholar
  37. Katzenellenbogen, J.A., Johnson, Jr., H.J., Carlson, K.E., and Myers, H.N., 1974, Photoreactivity of some light-sensitive estrogen derivatives. Use of an exchange assay to determine their photointeraction with the rat uterine estrogen binding protein, Biochemistry 13: 2986–2994.PubMedCrossRefGoogle Scholar
  38. Klip, A., and Gitler, C., 1974, Photoactive covalent labeling of membrane components from within the lipid core, Biochem. Biophys. Res. Commun. 60: 1155–1162.PubMedCrossRefGoogle Scholar
  39. Knowles, J.R., 1972, Photogenerated reagents for biological receptor-site labeling, Accounts Chem. Res. 5: 155–160.CrossRefGoogle Scholar
  40. Lifter, J., Hew, C.L., Yoshioka, M., Richards, F.F., and Konigsberg, W.H., 1974, Affinity-labeled peptides obtained from the combining region of myeloma protein 460. I. Heavy-chain-labeling patterns using dinitrophenyl azide photoaffinity label, Biochemistry 13: 3567–3571.PubMedCrossRefGoogle Scholar
  41. Lin, S.-Y, and Riggs, A.D., 1974, Photochemical attachment of lac repressor to bromodeoxyuridine-substituted lac operator by ultraviolet irradiation, Proc. Nat. Acad. Sci. USA, 71: 947–951.PubMedCrossRefGoogle Scholar
  42. Maasen, J.A., and Möller, W., 1974, Identification by photo-affinity labeling of the proteins in Escherichia coli ribosomes involved in elongation factor G-dependent GDP binding, Proc. Nat. Acad. Sci. USA 71: 1277–1280.CrossRefGoogle Scholar
  43. Markovitz, A., 1972, Ultraviolet light-induced stable complexes of DNA and DNA polymerase, Biochim. Biophys. Acta 281: 523–534.Google Scholar
  44. Martyr, R.J., and Benisek, W.F., 1973, Affinity labeling of the active sites o5-ketosteroid isomerase using photoexcited natural ligands, Biochemistry 12: 2172–2178.PubMedCrossRefGoogle Scholar
  45. Morrison, H.A., 1969, The photochemistry of the nitro and nitroso groups in “The Chemistry of the Nitro and Nitroso Groups,” (H. Feurer, ed.). pp. 165–213, Interscience, New York.Google Scholar
  46. Nakashima, Y., and Konigsberg, W., 1975, Photo-induced cross-linkage of gene 5 protein and bacteriophage fd DNA, Abstr. Int. Symp. “Protein and Other Adducts to DNA: Their Significance to Aging, Carcinogenesis and Radiation Biology,” Williamsburg, Virginia, May 2–6, 1975.Google Scholar
  47. Ogata, R., 1975, Affinity labeling of the operator binding site on the lac repressor, Abstr. Int. Symp. “Protein and Other Adducts to DNA: Their Significance to Aging, Carcinogenesis and Radiation Biology,” Williamsburg, Virginia, May 2–6, 1975.Google Scholar
  48. Richards, F.F., Lifter, J., Hew, C.L., Yoshioka, M., and Konigsberg, W.H., 1974, Photoaffinity labeling of the combining region of myeloma protein 460. II. An interpretation of the labeling patterns, Biochemistry 13: 3572–3575.PubMedCrossRefGoogle Scholar
  49. Rosenstein, R.W., and Richards, F.F., 1972, Synthesis of a photoactivate menadione (VIT K.). Affinity label in its reaction with a menadione-binding myeloma protein, J. Immunol. 108: 1467–1469.Google Scholar
  50. Rudnick, G., Kaback, H.R., and Wiel, R., 1975, Photoinactivation of the 8-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-ß-dgalactopyranoside, J. Biol. Chem. 250: 1371–1375.PubMedGoogle Scholar
  51. Ruoho, A.E., Kiefer, H., Roeder, P.E., and Singer, S.J., 1973, The mechanism of photoaffinity labeling, Proc. Nat. Acad. Sci. USA, 70: 2567–2571.PubMedCrossRefGoogle Scholar
  52. Ruoho, A., and Kyte, J.,1974 Photoaffinity labeling of the ouabain-binding site on (Na+ + K) adenosinetriphosphatase, Proc. Nat. Acad. Sci. USA, 71: 2352–2356.PubMedCrossRefGoogle Scholar
  53. Sawada, F., 1974, Kinetics of 4-thiouridylate-sensitized photoinactivation of ribonuclease A, Photochem, Photobiol. 20: 523–526.CrossRefGoogle Scholar
  54. Schenkman, M., Ward, D.C., and Moore, P.B. 1974, Covalent attachment of a messenger RNA to the Escherichia coli ribosome, Biochim. Biophys. Acta, 353: 503–508.PubMedCrossRefGoogle Scholar
  55. Schoemaker, H.J., and Schimmel, P.R., 1974, Photo-induced joining of a transfer RNA with its cognate amino acyl-transfer RNA synthetase, J. Mo Z. BioZ. 84: 503–513.CrossRefGoogle Scholar
  56. Schwartz, I., and Ofengand, J., 1974, Photo-affinity labeling of tRNA binding sites in macromolecules. I. Linking of the phenacyl-p-azide of 4-thiouridine in (Escherichia coli) valyltRNA to 16S RNA at the ribosomal P site, Proc. Nat. Acad. Sci. USA 71: 3951–3955.PubMedCrossRefGoogle Scholar
  57. Shafer, J., Baronowsky, P., Laursen, R., Finn, F., and Westheimer, F.H., 1966, Products from the photolysis of diazoacetyl chymotrypsin, J. Biol. Chem. 241: 421–427.PubMedGoogle Scholar
  58. Shaw, E., 1970, Chemical modification by active-site directed reagents, Enzymes 1: 91–146.CrossRefGoogle Scholar
  59. Singer, S.J., 1967, Covalent labeling of active sites, Adv. Protein Chem. 22: 1–54.PubMedCrossRefGoogle Scholar
  60. Singh, A., Thornton, E.R., and Westheimer, F.H., 1962, The photolysis of diazoacetyl chymotrypsin, J. Biol. Chem. 237:PC 3006.PubMedGoogle Scholar
  61. Smith, K.C., 1975, The radiation-induced addition of proteins and other molecules to nucleic acids, in “Photochemistry and Photobiology of Nucleic Acids” (S.Y. Wang, ed.), Academic Press, New York (in press).Google Scholar
  62. Smith, P.A.S., 1970, Aryl nitrenes and formation of nitrenes by rupture of heterocyclic rings, in “Nitrenes” (W. Lwowski, ed.), pp. 99–162, Wiley-Interscience, New York.Google Scholar
  63. Smith, R.A.G., and Knowles, J.R., 1974, The utility of photo-affinity labels as ‘mapping’ reagents. A study of sub-populations of a specific rabbit antibody by using structually related photo-affinity reagents, Biochem. J. 141: 51–56.PubMedGoogle Scholar
  64. Sonenberg, N., Zamir, A., and Wilchek, M., 1974, A photo induced reaction of chloramphenicol with E. coli ribosomes: covalent binding of the antibiotic and inactivation of peptidyl transferase, Biochem. Biophys. Res. Commun. 59: 693–696.PubMedCrossRefGoogle Scholar
  65. Staros, J.V., and Richards, F.M., 1974, Photochemical labeling of the surface proteins of human erythrocytes, Biochemistry 13: 2720–2726.PubMedCrossRefGoogle Scholar
  66. Staros, J.V., Haley, B.E., and Richards, F.M., 1974, Human erythrocytes and resealed ghosts, a comparison of membrane topology, J. Biol. Chem. 249: 5004–5007.PubMedGoogle Scholar
  67. Strniste, G.F., and Smith, D.A., 1974, Induction of stable linkage between the deoxyribonucleic acid dependent ribonucleic acid polymerase and d(A-T). d(A-T)n by ultraviolet light, Biochemistry 13:485–493.PubMedCrossRefGoogle Scholar
  68. Turro, N.J., Dalton, C.J., Dawes, R., Farrington, G., Harltala, R., Morton, D., Niemczyk, M., and Schore, N., 1972, Molecular photochemistry of alkanones in solution: a-cleavage, hydrogen abstraction, cycloaddition, and sensitization reactions, Accounts Chem. Res. 5: 92–101.CrossRefGoogle Scholar
  69. Vallee, B.L., and Riordan, J.F., 1969, Chemical approaches to the properties of active sites of enzymes, Annu. Rev. Biochem. 38: 733–794.PubMedCrossRefGoogle Scholar
  70. Vaughan, R., 1970, Diazo esters as bifunctional inhibitors. A method for marking the hydropholic binding sites of enzymes, Ph.D. Thesis, Harvard University.Google Scholar
  71. Vortek, P., 1975, Enhanced photoinactivation of thymidine phosphorylase by halogenated reactants accompanied by changes in physical properties of the enzyme. Abstr. Int. Symp. “Protein and Other Adducts to DNA: Their Significance to Aging, Carcinogenesis and Radiation Biology,” Williamsburg, Virginia, May 2–6, 1975.Google Scholar
  72. Wagner, P.J., 1971, Type II. Photoelimination and photocyclization of ketones, Accounts Chem. Res. 4: 168–177.CrossRefGoogle Scholar
  73. Walsh, C.T., Schonbrunn, A., Lockridge, O., Massey, V., and Abeles, R.H., 1972, Inactivation of a flavoprotein, lactate oxidase, by an acetylenic substrate, J. Biol. Chem. 247: 6004–6006.PubMedGoogle Scholar
  74. Weintraub, H., 1974, The assembly of newly replicated DNA into chromatin, CoZd Spring Harbor Symp. Quant. BioZ. 38: 247–256.CrossRefGoogle Scholar
  75. Wolff, M.E., Feldman, D., Catsoulacos, P., Funder, J.W., Hancock, C., Amano, Y., and Edelman, I.S., 1975, Steroidal 21-diazo ketones: photogenerated corticosteroid receptor labels, Biochemistry 14: 1750–1759.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media New York 1976

Authors and Affiliations

  • Barry S. Cooperman
    • 1
  1. 1.Department of ChemistryUniversity of PennsylvaniaPhiladelphiaUSA

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