Localization of the α-chain cross link acceptor sites of human fibrin

L. J. Fretto, E. W. Ferguson, H. M. Steinman, P. A. McKee

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36 Scopus citations


The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent α-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, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six α-chain peptides of molecular weight 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal α-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent α-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal α-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aα-chain. Cleavage of the MDC-labeled α-chain by CNBr, however, localized most of its fluorescence (~80%) to a fragment of molecular weight 29,000 which had the same NH2-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 NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aα-chain.

Original languageEnglish (US)
Pages (from-to)2184-2195
Number of pages12
JournalJournal of Biological Chemistry
Issue number7
StatePublished - 1978
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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