Relative Labilities of the Two Types of Interchain Disulfide Bond of Rabbit γG-Immunoglobulin

Abstract

R. HONG AND A. NISONOFF~ From the Department of Microbiology, University of Illinois, Urbana, Illinois (Received for publication, May 7, 1965) A molecule of rabbit yG-globulin’ evidently consists of four polypeptide chains held together by interchain disulfide bonds and noncovalent interactions. Each molecule comprises a pair of similar or identical “light” chains having an approximate molecular weight of 20,000, and another pair of “heavy” chains of weight 50,000 to 55,000 (2-7). Each light chain is linked to a heavy chain and the two heavy chains are joined to one another (3, 8). The presence of 1 X-carboxymethylcysteine residue per light chain, isolated after reduction and alkylation with iodoace- tate, indicates that a single interchain disulfide bond joins each light chain to a heavy chain (3). Similarly, in a large part if not all of the molecules, reduction of a single disulfide bond is sufficient to permit separation at 10~ pH of half-molecules, each consisting of a light and a heavy chain (9, 10). This is evidently the same disulfide bond that links two “univalent” (Fab’ (1)) fragments after peptic digestion (11). To be consistent with the structural model of Fleischman, Porter, and Press (3), it was necessary to postulate that this bond joins two heavy chains (11). The present investigation provides direct evidence that this is the case. A study was also made of the relative labilities of the disulfide bonds linking light and heavy chains as compared with that of the bond joining half-molecules.2 Conditions for preferential reduction of the H-H disulfide bond, with minimal cleavage of L-H disulfide bonds, were defined. The extent of exchange of light and heavy chains, under the conditions used for separation and hybridization of half-molecules, was estimated. Also, it was found that after reduction of all interchain disulfide bonds, separation into half-molecule subunits occurs under milder conditions than are required for dissociation into separate light and heavy chains.