Stepwise cleavage of rabbit immunoglobin G by papain and isolation of four types of biologically active Fc fragments

Abstract

Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ;hinge' region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ;hinge' region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5.0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4.5. Only the C-terminal antigenic determinants were retained by stFc fragment.

Induction of rheumatoid factors in mice by immune complexes of bacterial lipopolysaccharide with mouse IgG antibody

“…The quantitative reactivity of the serum with F(acb)z which possesses Cy2 but lacks Cy3 and its failure to react with Fab and the C,3 fragment indicated that RF possessed a restricted specificity to the Cy2 domain. The reactivity with mFc which lacks the hinge region [18] was decreased as compared to that with 1Fc carrying this region. Similar results were obtained by analysis of 25 individual sera from mice stimulated with IgG-TNP-LPS.…”

“…The pepsinderived C,3 fragment (Pep-111') [17], papain-derived Fab, lFc, mFc [18], and plasmin-derived F(acb)z [19] from RIgG were prepared as described. Identification of mFc and 1Fc was made by their dissociability and non-dissociability, respectively, in polyacrylamide gel electrophoresis in 0.1% SDS.…”

Structure and function of the constant regions of immunoglobulins

“…Non-covalently linked fragments consisting of V L dimer ) and V L and V H (Hochman, Inbar & Givol, 1973) have been prepared. Dimeric fragments consisting of most of the C y 3 domain have also been reported (Turner & Bennich, 1968;Utsumi, 1969;Ellerson et al 1972). On the other hand, Percy & Dorrington (1974) found that a light chain, from which most of the V domain was deleted, had no tendency to dimerize.…”

Partial Purification of Detergent-Soluble HL-A Antigen and Its Cleavage by Papain

“…This fact could be related to the finding of papain cleavage at more than one site. The analogous cleavage of immunoglobulins by papain yielding Fab and Fc products has been reported to occur at two sites in the hinge region of most immunoglobulins (34,35).…”