Advertisement

Chemical Strategy for Studying the Antigenic Structures of Disulfide-Containing Proteins: Hen Egg-White Lysozyme as a Model

  • M. Z. Atassi
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 86A)

Abstract

A critical approach in studying the immunochemistry of proteins relies on the preparation of a variety of long and overlapping peptides from the parent molecule. Disulfide-containing proteins are usually inaccessible to proteolytic attack and therefore it is not possible to prepare from such proteins a variety of peptides without rupturing the disulfides. Unfortunately, the preparations with broken disulfides bear no immunochemical relationship to the native proteins. These have until recently virtually eluded investigation. to break the deadlock, we introduced a novel cleavage approach for obtaining peptides from the native protein without rupturing the disulfide bonds. Reversible citraconylation of the amino groups induced conformational changes in the protein that were sufficient to render it completely accessible to tryptic hydrolysis at the arginyl bonds. Complete cleavage at the lysyl bonds may be effected, if desired, after deprotection of the amino groups which proceeds quantitatively. by this approach we obtained peptides from lysozyme which accounted for almost all (907%) of the immune reaction of native lysozyme. From these and the immunochemistry of numerous chemical derivatives of lysozyme, it was possible to derive the location of the antigenic sites of lysozyme. Two antigenic sites were then accurately delineated. One was achieved by organic synthesis of 9 disulfide peptides corresponding to various overlaps around the disulfide 6–127. for the delineation of the second site (around the disulfides 64–80 and 76–94), we devised (Atassi, Lee and Pai, 1976, Biochim. Biophys. Acta, 427, 745) an entirely novel “Surface-Simulation” synthetic approach. in this, the conformationally-adjacent residues constructing the site were linked directly via peptide bonds into a single peptide which does not exist in native lysozyme but simulates a surface region of it. This unorthodox approach provides a novel and powerful technique for final delineation of antigenic reactive sites (and perhaps other types of binding sites) in native proteins.

Keywords

Disulfide Bond Antigenic Site Antigenic Structure Immunochemical Reactivity Disulfide Reducibility 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Arnon, R., and Sela, M. (1969) Proc. Natl. Acad. Sci. USA 62:163PubMedCrossRefGoogle Scholar
  2. Arnon, R., Maron, E., Sela, M., and Anfinsen, C.B. (1971) Proc. Natl. Acad. Sci. USA 63:1450CrossRefGoogle Scholar
  3. Atassi, M.Z. (1968) Biochemistry 7:3078PubMedCrossRefGoogle Scholar
  4. Atassi, M.Z. (1969) Immunochemistry 6:801PubMedCrossRefGoogle Scholar
  5. Atassi, M.Z. (1972) Specific Receptors of Antibodies, Antigens and Cells, 118–136. 3rd International Convocation of Immunology, June 12–15, S. KargerGoogle Scholar
  6. Atassi, M.Z. (1975) Immunochemi s try 12:423CrossRefGoogle Scholar
  7. Atassi, M.Z. (1976) Immunochemistry of Proteins, p.1-161, Plenum.Google Scholar
  8. Atassi, M.Z. (1977) Immunochemistry of Proteins, Volume 2, in press, Plenum.Google Scholar
  9. Atassi, M.Z., and Habeeb, A.F.S.A. (1969) Biochemistry 8:1385PubMedCrossRefGoogle Scholar
  10. Atassi, M.Z., and Habeeb, A.F.S.A. (1972) Methods in Enzymology 25B:546Google Scholar
  11. Atassi, M.Z., and Habeeb, A.F.S.A. (1977) Immunochemistry of Proteins Volume 2, in press, Plenum.Google Scholar
  12. Atassi, M.Z. and Koketsu, J. (1975) Immunochemi s try 12:741CrossRefGoogle Scholar
  13. Atassi, M.Z., and Pai, R.C. (1975) Immunochemi s try 12:735CrossRefGoogle Scholar
  14. Atassi, M.Z. and Rosemblatt, M.C. (1974) J. Biol. Chem. 249:482Google Scholar
  15. Atassi, M.Z. and Saplin, B.J. (1968) Biochemistry 7:688PubMedCrossRefGoogle Scholar
  16. Atassi, M.Z., and Thomas, A.V. (1969) Biochemistry 8:3385PubMedCrossRefGoogle Scholar
  17. Atassi, M.Z., and Zablocki, W. (1975) Biochim. Biophys. Acta 386:233PubMedGoogle Scholar
  18. Atassi, M.Z., and Zablocki, W. (1976) J. Biol. Chem. 251:1653PubMedGoogle Scholar
  19. Atassi, M.Z., Perlstein, M.T. and Habeeb, A.F.S.A. (1971) J. Biol. Chem. 246:4291Google Scholar
  20. Atassi, M.Z., Suliman, A.M., and Habeeb, A.F.S.A. (1972) Immimochemistry 9:907CrossRefGoogle Scholar
  21. Atassi, M.Z., Habeeb, A.F.S.A., and Ando, K. (1973) Biochim. Biophys. Acta 303:203PubMedGoogle Scholar
  22. Atassi, M.Z., Rosemblatt, M.C., and Habeeb, A.F.S.A. (1974) Immunochemistry 11:495PubMedCrossRefGoogle Scholar
  23. Atassi, M.Z., Suliman, A.M., and Habeeb, A.F.S.A. (1975a) Biochim. Biophys. Acta 405:452PubMedGoogle Scholar
  24. Atassi, M. Z., Habeeb, A.F.S.A., and Lee, C.L. (1976a) Immunochemistry, acceptedGoogle Scholar
  25. Atassi, M.Z., Koketsu, J., and Habeeb, A.F.S.A. (1976b) Biochim. Biophys. Acta 420:358PubMedGoogle Scholar
  26. Atassi, M.Z., Lee, C.L., and Habeeb, A.F.S.A. (1976c) Immunochemistry 13:7PubMedCrossRefGoogle Scholar
  27. Atassi, M.Z., Lee, C.L., and Pai, R.C. (1976d) Biochim. Biophys. Acta 427:745PubMedGoogle Scholar
  28. Blake, C.C.F., Koenig, D.F., Mair, G.A., North, A.C.T., Phillips, D.C., and Sarma, V.R. (1965) Nature (Lond) 206:757CrossRefGoogle Scholar
  29. Bonavida, B., Miller, A., and Sercarz, E.E. (1969) Biochemistry 8:968PubMedCrossRefGoogle Scholar
  30. Brown, R.K. (1962) J. Biol. Chem. 238:1162Google Scholar
  31. Brown, R.K., Durieux, J., Delaney, R., Leikem, E., and Clark, B.J. (1959) Ann. N.Y. Acad. Sci. 81:524PubMedCrossRefGoogle Scholar
  32. Canfield, R.E. (1963a) J. Biol. Chem. 238:2691PubMedGoogle Scholar
  33. Canfield, R.E. (1963b) J. Biol. Chem. 238:2698PubMedGoogle Scholar
  34. Canfield, R.E., and Liu, A.K. (1965) J. Biol. Chem. 240:1997PubMedGoogle Scholar
  35. Edelhoch, H. (1962) J. Biol. Chem. 237:2778Google Scholar
  36. Fujio, H., Imanishi, M., Nishioka, K., and Amano, T. (1968a) Biken J. 11:207Google Scholar
  37. Fujio, H., Imanishi, M., Nishioka, K., and Amano, T. (1968b) Biken J. 11:219PubMedGoogle Scholar
  38. Fujio, H., Martin, R.E., Ha, Y.M., Sakato, N., and Amano, T. (1974) Biken J. 17:73PubMedGoogle Scholar
  39. Geiger, B., and Arnon, R. (1974) Eur. J. Immunol. 4:632PubMedCrossRefGoogle Scholar
  40. Gerwing, J., and Thompson, K. (1968) Biochemistry 7:3888PubMedCrossRefGoogle Scholar
  41. Ha, Y.M., Fujio, H., Sakato, N., and Amano, T. (1975) Biken J. 18:47PubMedGoogle Scholar
  42. Habeeb, A.F.S.A., and Atassi, M.Z. (1969) Immunochemistry 6:555PubMedCrossRefGoogle Scholar
  43. Habeeb, A.F.S.A., and Atassi, M.Z. (1970) Biochemistry 9:4939PubMedCrossRefGoogle Scholar
  44. Habeeb, A.F.S.A., and Atassi, M.Z. (1971b) Immunochemistry 8:1047PubMedCrossRefGoogle Scholar
  45. Habeeb, A.F.S.A., Atassi, M.Z., and Lee, C.L. (1974) Biochim. Biophys. Acta 342:389PubMedGoogle Scholar
  46. Imoto, T., Johnson, L.N., North, A.C.T., Phillips, D.C., and Rupley, J. A. (1972) The Enzymes (Boyer, P.D., Ed.) 7:665, Academic Press, N.Y.Google Scholar
  47. Johnson, E.R., Anderson, W.L., Wetlaufer, D.B., Lee, C.L., and Atassi, M.Z. (1976) J. Biol. Chem., submittedGoogle Scholar
  48. Jolies, J., Jauregui-Adell, J., Bernier, I., and Jolies, P. (1963) Biochim. Biophys. Acta 78:668CrossRefGoogle Scholar
  49. Jolies, J., Sportono, G., and Jolies, P. (1965) Nature (Lond) 208:1204CrossRefGoogle Scholar
  50. Jolies, P., Jauregui-Adell, J., and Jolies, J. (1964) Compt. Rend. Acad. Sci. 258:3926Google Scholar
  51. Koketsu, J., and Atassi, M.Z. (1973) Biochim. Biophys. Acta 328:289PubMedGoogle Scholar
  52. Koketsu, J., and Atassi, M.Z. (1974a) Immunochemistry 11:1PubMedCrossRefGoogle Scholar
  53. Koketsu, J., and Atassi, M.Z. (1974b) Biochim. Biophys. Acta 342:21PubMedGoogle Scholar
  54. Komatsu, T., Shinka, S., Dohi, Y., and Amano, T. (1975) Biken J. 18:61PubMedGoogle Scholar
  55. Lee, C.L., and Atassi, M.Z. (1973) Biochemistry 12:2690PubMedCrossRefGoogle Scholar
  56. Lee, C.L., and Atassi, M.Z. (1975) Biochim. Biophys. Acta 405:464PubMedGoogle Scholar
  57. Lee, C.L., and Atassi, M.Z. (1976) Biochem. J. 159:89PubMedGoogle Scholar
  58. Lee, C.L., Atassi, M.Z., and Habeeb, A.F.S.A. (1975) Biochim. Biophys. Acta 400:423Google Scholar
  59. Lee, C.L., Pai, R.C., and Atassi, M.Z. (1976) Immunochemistry 13:681PubMedCrossRefGoogle Scholar
  60. Maron, E., Shiozawa, C., Arnon, R., and Sela, M. (1971) Biochemistry 10:763PubMedCrossRefGoogle Scholar
  61. Sakato, N., Fujio, H., and Amano, T. (1972) Biken J. 15:135PubMedGoogle Scholar
  62. Shinka, S., Imanishi, M., Miyagawa, N., Amano, T., Inouye, M., and Tsugita, A. (1967) Biken J. 10:89PubMedGoogle Scholar
  63. Shrake, A., and Rupley, J.A. (1973) J. Mol. Biol. 79:351PubMedCrossRefGoogle Scholar
  64. Singhai, R.P., and Atassi, M.Z. (1971) Biochemistry 10:1756CrossRefGoogle Scholar
  65. Sokolovsky, M., Riordan, J.F., and Vallee, B.L. (1967) Biochem. Biophys. Res. Commun. 27:20PubMedCrossRefGoogle Scholar
  66. Strosberg, A.D., and Kanarek, L. (1970) Eur. J. Biochem. 14:161PubMedCrossRefGoogle Scholar
  67. Taubman, M.T., and Atassi, M.Z. (1968) Biochem. J. 106:829PubMedGoogle Scholar
  68. Young, J.D., and Leung, C.Y. (1970) Biochemistry 9:2755PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1977

Authors and Affiliations

  • M. Z. Atassi
    • 1
    • 2
  1. 1.Department of ImmunologyMayo Medical SchoolRochesterUSA
  2. 2.Department of BiochemistryUniversity of MinnesotaMinneapolisUSA

Personalised recommendations