Localization of the a-Chain Cross-link Acceptor Sites of Human Fibrin*

The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent a-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH,-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six a-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH,-terminal a-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sites which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent a-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH,-terminal a-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH,-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aa-chain. Cleavage of the MDC-labeled a-chain by CNBr, however, localized most of its fluorescence (-80%) to a fragment of molecular weight 29,000 which had the same NH,-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH,-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites