IMMUNOCHEMISTRY OF SPERM WHALE MYOGLOBIN .4. ROLE OF ARGININE RESIDUES IN CONFORMATION AND DIFFERENTIATION OF THEIR ROLES IN ANTIGENIC REACTIVITY

Reaction of apomyoglobin with cyclohexanedione has been investigated. Modified apomyoglobin was recombined with ferriheme and the metmyoglobin obtained was subjected to chromatography on CM-cellulose. A homogeneous fraction was isolated which was modified at three arginine residues. The modified arginines were located at positions 31, 118, and 139. The derivative possessed spectral and hydrodynamic parameters identical with those of metmyoglobin X. With antisera to metymyoglobin X it reacted less than the homologous antigen. Apomyoglobin prepared from this metmyoglobin derivative also reacted less than unmodified apomyoglobin. Fragments obtained by cleavage of apomyoglobin atmethionines-55 and -131 were also modified with cyclohexanedione. The immunochemical reaction of peptide 1-55 was unaffected upon modification of arginines 31 and 45. Similarly the inhibitory activity of peptide 132-153 did not change after modification of arginine-139. On the other hand, modification of arginine-118 in peptide 56-131 resulted in an appreciable decrease of the immunochemical reactivity of this peptide. This decrease in the reaction of the modified peptide accounted for the entire loss of immune reaction obtained with modified apomyoglobin and almost all that obtained with modified metmyoglobin. These findings suggest that of the four arginine residues in metmyoglobin, only arginine- 118 is located in an antigenic reactive region. The results also show that the environment of an amino acid type determines its involvement in the antigenic structure of a globular protein. The present data have been integrated with previous results concerning the antigenic structure of metmyoglobin. © 1969, American Chemical Society. All rights reserved.