Selective reduction and proteolysis in the hinge region of liganded and unliganded antibodies. Identical kinetics suggest lack of major conformational change in the hinge region

Abstract The experiments described here were designed to test the validity of the allosteric immunoglobulin model of Huber et al. ( Nature 1976. 264 : 415) according to which antigen could cause a stiffening of the flexible antibody molecule by the formation of longitudinal CH 1 –CH 2 interdomain contacts. The largest structural change would involve the folding of the hinge peptide in‐between the CH 2 globules. Consequently, the accessibility of the hinge region to reduction and limited proteolysis should be considerably altered. Homogeneous rabbit antibodies to type II and III pneumococcal polysaccharide were reacted with corresponding oligosaccharide haptens and whole polysaccharide antigens to form monomeric and polymeric IgG antibody complexes, respectively. Reduction of the inter‐heavy chain disulfide bond in the hinge region by a series of thiol reagents of increasing size and polarity was performed; the ratio of reduction rate in complexes with hapten or antigen compared to that of hapten‐or antigen‐free control was found to be close to unity, thus indicating that the susceptibility of the hinge disulfide bond was unaffected by the binding of hapten or antigen. Limited proteolysis in the hinge region of immune complexes by activator‐free papain or pepsin in the absence of reducing agent was carried out, and the appearance of antibody fragments followed by sodium dodecyl sulfate gel electrophoresis. The rate of proteolysis in hapten‐antibody complexes and in large soluble antigen‐antibody aggregates (16–29 S) did not show any significant decrease when compared to that for unliganded antibody. The identity of the reduction and proteolysis rates in the presence and absence of antigen provides strong evidence for the lack of a major conformational change in the hinge region upon antigen binding.