Enzymic and immunochemical properties of lysozyme. XI. Conformation and immunochemistry of the two-disulfide peptide and the tryptophan and lysine residues in its antigenic reactivity.
The previously described peptide 62-68 (Cys 64-Cys 80) 74-96 (Cys 76-Cys 94) (Atassi, M.Z., Suliman, A.M. and Habeeb, A.F.S.A. (1975) Biochim. Biophys. Acta 405, 452-463), which accounted for about one-third of the total antigenic reactivity of native lysozyme, was isolated here with lysine 97 attached to it. The peptide was subjected to specific modification reactions in order to determine some of the residues which formed part of its antigenic reactive site. ORD measurements showed that the peptide was greatly unfolded in solution relative to its expected mode of folding within the intact lysozyme molecule. Modification of the two tryptophan residues in the peptide by reaction with 2,3-dioxo-5-indolinesulfonic acid provided a derivative which possessed similar conformational parameters to those of the unmodified peptide. However, the derivative retained only about half the immunochemical reactivity of the peptide. Succinylation of the amino groups afforded a derivative whose conformational parameters were identical to those of the unmodified peptide but in which half of the immunochemical reactivity was lost. Modification of the two tryptophan residues followed by succinylation of the amino groups resulted in almost complete loss of the antigenic reactivity, and the loss was not due to conformational differences. The antigenic reactivity of the peptide was also destroyed on removal of tryptophans 62 and 63, of sequence 84-93 from the loop 74-79 and of sequence 74-75 by chymotryptic digestion. From these and previous results it was concluded that the antigenic reactive site in this part of the lysozyme molecule incorporates one or both of tryptophans 62 and 63 as well as one or both lysines 96 and 97. The two disulfides 64-80 and 76-94 bring these two parts of the lysozyme molecule into a single reactive site. The intactness of the disulfides is essential for maintenance and reactivity of the site.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004220:Disulfides / Q000032:analysis; D004529:Egg White; D007211:Indoles; D008239:Lysine / Q000032:analysis; D008970:Molecular Weight; D009113:Muramidase / Q000276:immunology / Q000378:metabolism; D009905:Optical Rotatory Dispersion; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D013386:Succinates; D014357:Trypsin; D014364:Tryptophan / Q000032:analysis
D000596:Amino Acids; D004220:Disulfides; D007211:Indoles; D010446:Peptide Fragments; D013386:Succinates; D014364:Tryptophan; D009113:Muramidase; D014357:Trypsin; D008239:Lysine