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. 1978 May 1;171(2):419–427. doi: 10.1042/bj1710419

Boundary refinement of the lysozyme antigenic site around the disulphide bond 6–127 (site 1) by `surface-simulation' synthesis*

M Zouhair Atassi 1,†, Ching-Li Lee 1
PMCID: PMC1183971  PMID: 656053

Abstract

1. We have previously shown that an antigenic site (site 1) in native lysozyme resides around the disulphide bond 6–127 and, by classical synthesis of nine disulphide peptides, the antigenic site was accurately narrowed down to the structure Cys(6)–Arg(14)–[Cys(6)–Cys(127)] –Gly(126)–Arg(128). Only a few residues on this disulphide peptide were proposed to be involved in the reactivity with antibody. However, this lacked direct verification and the role of Arg-128 remained uncertain. 2. In the present work, several peptides were designed and synthesized by the surface-simulation concept devised in our laboratory. These enabled the precise definition of the site as well as the investigation of its conformational and directional requirements. 3. The results showed that the antigenic site (site 1) is made up of the spatially contiguous surface residues: Arg-125, Arg-5, Glu-7, Arg-14, Lys-13. The surface-simulation synthetic peptide Arg-Gly-Gly-Arg-Gly-Glu-Gly-Gly-Arg-Lys (which does not exist in native lysozyme, but copies a surface region of it) accounted entirely for the maximum expected reactivity of the site (i.e. about one-third of the total antigenic reactivity of lysozyme). An immunoadsorbent of the peptide also removed about one-third of the total lysozyme antibodies. 4. The antigenic site exhibited restricted conformational freedom. The achievement of the full reactivity of the site by surface-simulation synthesis requires the appropriate choice of spacer separation between its reactive residues. The surface-simulation synthetic site exhibits the same mono-directional preference (Arg-125 to Lys-13) for the rabbit and goat antisera so far tested. The site describes a line which encircles a part (3.01 nm in C(α)-to-C(α) distance from Arg-125 to Lys-13) of the surface of the molecule.

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Selected References

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  1. Atassi M. Z. Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins. Immunochemistry. 1975 May;12(5):423–438. doi: 10.1016/0019-2791(75)90010-5. [DOI] [PubMed] [Google Scholar]
  2. Atassi M. Z. Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins. Immunochemistry. 1975 May;12(5):423–438. doi: 10.1016/0019-2791(75)90010-5. [DOI] [PubMed] [Google Scholar]
  3. Atassi M. Z., Habeeb A. F., Ando K. Enzymic and immunochemical properties of lysozyme. VII. Location of all the antigenic reactive regions. A new approach to study immunochemistry of tight proteins. Biochim Biophys Acta. 1973 Mar 23;303(1):203–209. [PubMed] [Google Scholar]
  4. Atassi M. Z., Habeeb A. F., Lee C. L. Immunochemistry of serum albumin--II. Isolation and characterization of a fragment from the first third of bovine serum albumin carrying almost all the antigenic reactivity of the protein. Immunochemistry. 1976 Jun;13(6):547–555. doi: 10.1016/0019-2791(76)90332-3. [DOI] [PubMed] [Google Scholar]
  5. Atassi M. Z. Immunochemistry of sperm whale myoglobin. VI. Preparation and conformational analysis of eight mammalian myoglobins. Biochim Biophys Acta. 1970 Dec 22;221(3):612–622. doi: 10.1016/0005-2795(70)90233-3. [DOI] [PubMed] [Google Scholar]
  6. Atassi M. Z. Immunochemistry of sperm-whale myoglobins prepared with various modified porphyrins and metalloporphyrins. Biochem J. 1967 Apr;103(1):29–35. doi: 10.1042/bj1030029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Atassi M. Z., Koketsu J., Habeeb A. F. Enzymic and immunochemical properties of lysozyme. XIII. Accurate delineation of the reactive site around the disulfide 6-127 by immunochemical study of beta-propiolactone lysozyme derivative and of synthetic disulfide peptides. Biochim Biophys Acta. 1976 Feb 20;420(2):358–375. doi: 10.1016/0005-2795(76)90328-7. [DOI] [PubMed] [Google Scholar]
  8. Atassi M. Z., Koketsu J. Immunochemistry of sperm-whale myoglobin--XXIII. Investigation of the independence of the five antigenic reactive regions by immunoabsorbent studies. Immunochemistry. 1975 Sep;12(9):741–744. [PubMed] [Google Scholar]
  9. Atassi M. Z., Lee C. L., Pai R. C. Enzymic and immunochemical properties of lysozyme. XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site. Biochim Biophys Acta. 1976 Apr 14;427(2):745–751. doi: 10.1016/0005-2795(76)90219-1. [DOI] [PubMed] [Google Scholar]
  10. Atassi M. Z., Lee C. L. The precise and entire antigenic structure of native lysozyme. Biochem J. 1978 May 1;171(2):429–434. doi: 10.1042/bj1710429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Atassi M. Z., Li Lee C. Enzymic and immunochemical properties of lysozyme-XII. Delineation of the reactive site around the two central disulfides by immunochemical and conformational studies of derivatives of the two-disulfide peptide. Immunochemistry. 1976 Jan;13(1):7–14. doi: 10.1016/0019-2791(76)90290-1. [DOI] [PubMed] [Google Scholar]
  12. Atassi M. Z., Pai R. C. Immunochemistry of sperm-whale myoglobin--XXII. Accurate delineation of the single reactive region in sequence 103-120 by immunochemical studies of synthetic peptides: the complete antigenic structure of the protein. Immunochemistry. 1975 Sep;12(9):735–740. [PubMed] [Google Scholar]
  13. Atassi M. Z. Periodate oxidation of sperm-whale myoglobin and the role of the methionine residues in the antigen-antibody reaction. Biochem J. 1967 Feb;102(2):478–487. doi: 10.1042/bj1020478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Atassi M. Z., Perlstein M. T., Habeeb A. F. Conformational studies on modified proteins and peptides. IV. Conformation of lysozyme derivatives modified at tyrosine or at tryptophan residues. J Biol Chem. 1971 May 25;246(10):3291–3296. [PubMed] [Google Scholar]
  15. Atassi M. Z., Rosemblatt M. C., Habeeb A. F. Enzymic and immunochemical properties of lysozyme--VIII. Modification of two carboxyl groups and their role in the antigenic reactivity. Immunochemistry. 1974 Aug;11(8):495–500. doi: 10.1016/0019-2791(74)90121-9. [DOI] [PubMed] [Google Scholar]
  16. Atassi M. Z., Saplin B. J. Immunochemistry of sperm whale myoglobin. I. The specific interaction of some tryptic peptides and of peptides containing all the reactive regions of the antigen. Biochemistry. 1968 Feb;7(2):688–698. doi: 10.1021/bi00842a026. [DOI] [PubMed] [Google Scholar]
  17. Atassi M. Z., Suliman A. M., Habeeb A. F. Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups. Biochim Biophys Acta. 1975 Oct 20;405(2):452–463. doi: 10.1016/0005-2795(75)90110-5. [DOI] [PubMed] [Google Scholar]
  18. Atassi M. Z., Thomas A. V. Immunochemistry of sperm whale myoglobin. IV. The role of the arginine residues in the conformation and differentiation of their roles in the antigenic reactivity. Biochemistry. 1969 Aug;8(8):3385–3394. doi: 10.1021/bi00836a037. [DOI] [PubMed] [Google Scholar]
  19. Atassi M. Z., Zablocki W. 2,3-Dioxo-5-indolinesulfonic acid, a new highly specific reagent for modification of tryptophan in peptides and proteins. Biochim Biophys Acta. 1975 Mar 28;386(1):233–238. doi: 10.1016/0005-2795(75)90263-9. [DOI] [PubMed] [Google Scholar]
  20. Atassi M. Z., Zablocki W. Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid. J Biol Chem. 1976 Mar 25;251(6):1653–1658. [PubMed] [Google Scholar]
  21. Blake C. C., Mair G. A., North A. C., Phillips D. C., Sarma V. R. On the conformation of the hen egg-white lysozyme molecule. Proc R Soc Lond B Biol Sci. 1967 Apr 18;167(1009):365–377. doi: 10.1098/rspb.1967.0034. [DOI] [PubMed] [Google Scholar]
  22. Brandt J., Andersson L. O., Porath J. Covalent attachment of proteins to polysaccharide carriers by means of benzoquinone. Biochim Biophys Acta. 1975 Mar 28;386(1):196–202. doi: 10.1016/0005-2795(75)90259-7. [DOI] [PubMed] [Google Scholar]
  23. Habeeb A. F., Atassi M. Z. A fragment comprising the last third of bovine serum albumin which accounts for almost all the antigenic reactivity of the native protein. J Biol Chem. 1976 Aug 10;251(15):4616–4621. [PubMed] [Google Scholar]
  24. Habeeb A. F., Atassi M. Z. Enzymic and immunochemical properties of lysozyme. IV. Demonstration of conformational differences between alpha-lactalbumin and lysozyme. Biochim Biophys Acta. 1971 Apr 27;236(1):131–141. doi: 10.1016/0005-2795(71)90158-9. [DOI] [PubMed] [Google Scholar]
  25. Habeeb A. F., Atassi M. Z., Lee C. L. Peptides with strong immunochemical inhibitory activity from bovine serum albumin. Application of a novel approach. Biochim Biophys Acta. 1974 Apr 11;342(2):389–395. doi: 10.1016/0005-2795(74)90097-x. [DOI] [PubMed] [Google Scholar]
  26. Koketsu J., Atassi M. Z. Immunochemistry of sperm-whale myoglobin. 18. Accurate delineation of the single reactive region in sequence 120-153 by study of synthetic peptides. Biochim Biophys Acta. 1973 Dec 6;328(2):289–302. [PubMed] [Google Scholar]
  27. Koketsu J., Atassi M. Z. Immunochemistry of sperm-whale myoglobin. XIX. Accurate delineation of the single reactive region in sequence 54-85 by immunochemical study of synthetic peptides. Biochim Biophys Acta. 1974 Mar 14;342(1):21–29. [PubMed] [Google Scholar]
  28. Koketsu J., Atassi M. Z. Immunochemistry of sperm-whale myoglobin. XVI. Accurate delineation of the single region in sequence 1-55 by immunochemical studies of synthetic peptides. Some conclusions concerning antigenic structures of proteins. Immunochemistry. 1974 Jan;11(1):1–8. doi: 10.1016/0019-2791(74)90335-8. [DOI] [PubMed] [Google Scholar]
  29. Lee C. L., Atassi M. Z. Delineation of the third antigenic site of lysozyme by application of a novel 'surface-simulation' synthetic approach directly linking the conformationally adjacent residues forming the site. Biochem J. 1976 Oct 1;159(1):89–93. doi: 10.1042/bj1590089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee C. L., Atassi M. Z. Enzymic and immunochemical properties of lysozyme. Accurate definition of the antigenic site around the disulphide bridge 30-115 (site 3) by 'surface-simulation' synthesis. Biochem J. 1977 Dec 1;167(3):571–581. doi: 10.1042/bj1670571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee C. L., Atassi M. Z. Enzymic and immunochemical properties of lysozyme. XIX. Definition of the direction and conformational restrictions of the antigenic site around the disulfide bonds 64-80 and 76-94 (site 2) by 'surface-simulation' synthesis. Biochim Biophys Acta. 1977 Dec 20;495(2):354–368. doi: 10.1016/0005-2795(77)90391-9. [DOI] [PubMed] [Google Scholar]
  32. Lee C. L., Pai R. C., Atassi M. Z. Enzymic and immunochemical properties of lysozyme--XV. Delineation of the reactive site around the two central disulfides by immunochemical studies of novel synthetic peptides that contain diglycyl bridges instead of disulfides. Immunochemistry. 1976 Aug;13(8):681–687. doi: 10.1016/0019-2791(76)90209-3. [DOI] [PubMed] [Google Scholar]
  33. March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
  34. Pai R. C., Atassi M. Z. Immunochemistry of sperm-whale myoglobin--XX. Accurate delineation of the single reactive region in sequence 80-103 by immunochemical studies of synthetic peptides. Immunochemistry. 1975 Apr;12(4):285–290. doi: 10.1016/0019-2791(75)90177-9. [DOI] [PubMed] [Google Scholar]
  35. Trotta P. P., Estis L. F., Meister A., Haschemeyer R. H. Self-association and allosteric properties of glutamine-dependent carbamyl phosphate synthetase. Reversible dissociation to monomeric species. J Biol Chem. 1974 Jan 25;249(2):482–489. [PubMed] [Google Scholar]

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