THE COMPONENT FRAGMENTS OBTAINED BY ACID DISSOCIATION OF PAPAIN‐SOLUBILIZED H‐2 MOLECULES*

Abstract

H-2Kk and H-2Dd molecules were specificially purified from a radioiodinated H-2a preparation obtained by papain digestion of spleen cell membranes of A/J strain mice. The molecules were isolated by binding to H-2 alloantisera of the corresponding private specificity followed by precipitation with rabbit anti-mouse IgG antiserum. The specifically precipitated radioiodinated H-2Kk and H-2Dd molecules were dissociated by acid treatment into large and small components of about 37,000 and 11,000 respectively. These were separated by gel filtration at acid pH or by gel isoelectric focusing in the presence of 6 M urea. Each component separated by gel filtration of the acid-dissociated H-2 molecules showed a high degree of size homogeneity as determined by sodium dodecyl sulphate-acrylamide gel electrophoresis. Upon gel isoelectric focusing, however, the small components showed two peaks of radioactivity closely located together at pH 7-8, both of which had a restricted pH range, while the large components gave one peak of a relatively wide pH range of pH 5-6. The H-2Kk and H-2Dd molecules gave essentially the same pattern in terms of the numbers and the positions of the radioactivity bands. Under the iodination conditions used the large components of H-2Kk molecules contained more radioactivity than the small components, while the reverse was true in case of H-2Dd molecules. Such a difference was also found with H-2Kk and H-2Dd molecules isolated by use of alloantisera of the respective public specificity. The assay of binding of the isolated components with H-2 alloantisera of defined specificity revealed that the large components retain most of the allospecificities of the parental H-2 molecules. No H-2 allospecificities were found on the small components. The small components showed extensive binding with rabbit antiserum against mouse beta2-microglobulin. The same antiserum did not show any binding with the large components. On the other hand, both of the components did bind with rabbit antiserum against papain-solubilized H-2 molecules.