Aggregation of Fc′µ Subfragments Produced by Tryptic Hydrolysis of Human IgM at Elevated Temperature

[摘要] The proteolysis of Waldenstrom macroglobulins into Fabµ and (Fc)5µ fragments by tryptic digestion at 56°C to 65°C was reexamined. It appeared that “(Fc)5µ fragment” is composed of both Fc′µ subfragments and residual “(Fc)5µ fragment” held together by non-covalent forces.Sephadex G-200 chromatography resolved a 65°C tryptic digest of IgM into three peaks: GI, (Fc)5µ fragment; GII, Fabµ fragment; and peptides. Gel-filtration of an identical IgM digest through Sepharose 6B resulted in four peaks: BI, BII, BIII and peptides. BIII with m.w. 52,000 according to SDS polyacrylamide gel electrophoresis is Fabµ fragment. GI and BII, having sedimentation coefficients of 9.9S and 0.1S, respectively, were found to be antigenically equivalent according to Fcµ specific antisera. BI is apparently a high molecular weight aggregate of Fc′µ subfragments of 10,000 m.w. This aggregate was antigenically deficient in Fcµ determinants to both GI and BII. SDS gel electrophoresis showed that the appearance of BI subfragment was not dependent upon reduction and alkylation. This suggests that BI is derived from a region of the µ-chain other than the area containing inter-µ-chain disulfide bonds. Reduced-alkylated GI is characterized by major protein bands at m.w. 10,000, 12,500, 16,000, and 43,000 in SDS gel electrophoresis. BII, whose reduced-alkylated form is devoid of the band corresponding to m.w. 10,000, is most likely residual “(Fc)5µ fragment”.It appears that BI results from further fragmentation of (Fc)5µ fragment with subsequent aggregation. The release of BI is temperature dependent starting at 59°C and is greatest at 65°C. BI contains < 2% hexose and is presumably derived from a carbohydrate poor Cµ domain; BII contains 11% hexose. BI fraction was further purified by passage through a Sepharose 6B column equilibrated with 5 M guanidine HCl. Comparison of preliminary N- and C-terminal sequence data with published IgM sequence data shows BI subfragment to be located in the C-terminal region of Fcµ fragment.

[学科分类] 生物科学（综合）