IMMUNOCHEMISTRY OF SPERM WHALE MYOGLOBIN .I. SPECIFIC INTERACTION OF SOME TRYPTIC PEPTIDES AND OF PEPTIDES CONTAINING ALL REACTIVE REGIONS OF ANTIGEN

Sperm whale apomyoglobin was digested with trypsin and the soluble peptides were fractionated by chromatography. Five peptides, which occupied mostly corners in the three-dimensional structure of Kendrew et al. (Kendrew, J. C., Watson, H. C., Strandberg, B. E., Dickerson, R. E., Phillips, D. C., and Shore, V. C. (1961), Nature 190, 666) were purified and their immunochemical reactivities were studied. These peptides occupied in the parent protein the corners A-B (i.e., the bend between helices A and B in the three-dimensional model), E-F, F-G, and G-H and also most of helix B, all of helices F and C and the C-terminal hexapeptide. None of the peptides precipitated antibody and only inhibitory activities were detected. The greatest inhibitory activity resided in the segment A-B. This was followed, in degree of reactivity by segment E-F-G (i.e., corner between helices E and F, all of helix F and corner between helices F and G). The C-terminal hexapeptide was also reactive as was the segment comprising the bend G-H. The peptide corresponding to helix C showed no or very little inhibitory activity. It was therefore concluded that the foregoing corners on the surface of the molecule are present in antigenically active regions. Apomyoglobin was also subjected to cleavage at tryptophan-7 and/or at the two methionine sites. The shortened apopoprotein was immunochemically intact (relative to oxidized, uncleaved control). This, in addition to the finding that fragment 1-7 was inactive, suggested that the latter fragment is not part of an antigenic site in the intact molecule. There are indications, however, that sequence 1-7 might be important for the proper orientation of the reactive region(s) on fragment 1-55. Fragments 1-55 and 56-131 gave immune precipitates with antibodies to the whole protein. Fragment 132-153 showed only inhibitory activity with all the five sera tested. With a given antiserum, a greater portion of the reactivity appeared in the core fragment (i.e., 56-131). However, the three fragments obtained by cleavage at the methionines accounted for almost all the immunochemical reactivity of the intact antigen. The significance of these findings is discussed. © 1968, American Chemical Society. All rights reserved.