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

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

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

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
Volume253
Issue number7
StatePublished - 1978
Externally publishedYes

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Cadaverine
Fibrin
Peptides
Molecular Weight
Molecular weight
Cyanogen Bromide
Ancrod
Sequence Analysis
Digestion
Fluorescence
Disc Electrophoresis
Derivatives
Fibrinolysin
Protein Sequence Analysis
Sodium Dodecyl Sulfate
Disulfides
Electrophoresis
Chromatography
Gels
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Localization of the α-chain cross link acceptor sites of human fibrin. / Fretto, L. J.; Ferguson, E. W.; Steinman, Howard M.; McKee, P. A.

In: Journal of Biological Chemistry, Vol. 253, No. 7, 1978, p. 2184-2195.

Research output: Contribution to journalArticle

Fretto, LJ, Ferguson, EW, Steinman, HM & McKee, PA 1978, 'Localization of the α-chain cross link acceptor sites of human fibrin', Journal of Biological Chemistry, vol. 253, no. 7, pp. 2184-2195.
Fretto, L. J. ; Ferguson, E. W. ; Steinman, Howard M. ; McKee, P. A. / Localization of the α-chain cross link acceptor sites of human fibrin. In: Journal of Biological Chemistry. 1978 ; Vol. 253, No. 7. pp. 2184-2195.
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