Fretto L J, Ferguson E W, Steinman H M, McKee P A
J Biol Chem. 1978 Apr 10;253(7):2184-95.
The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-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 alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-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 alpha-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 Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 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 Aalpha-chain.
用荧光替代交联供体单丹磺酰尸胺（MDC）对纤维蛋白潜在的交联受体位点进行特异性标记。通过十二烷基硫酸钠圆盘凝胶电泳鉴定了几种由标记纤维蛋白经酶解和溴化氰（CNBr）裂解产生的荧光α链肽；将它们分离出来，然后通过氨基酸分析、氨基末端序列分析以及对其消化产物的色谱和电泳分析进行表征。Ancrod酶解MDC标记的纤维蛋白产生了一系列六种分子量在34,000至12,000之间的α链肽，每种肽都含有一个MDC标记的受体位点，以及一个分子量为37,500的氨基末端α链衍生物。后者通过二硫键结合在残留的纤维蛋白中，并且有两个可通过CNBr裂解分离的MDC标记位点。用温和的纤溶酶消化MDC标记的纤维蛋白，在上清液中产生了分子量为45,000、42,000、35,000、23,000、21,000和2,500的荧光α链肽，以及一个分子量为25,000的非荧光氨基末端α链衍生物，其保留在不溶性残留纤维蛋白中。通过氨基末端序列分析确定了这些纤溶酶上清液肽的排列顺序，结果表明一个MDC受体位点位于Aα链的大约第255位残基处。然而，用CNBr裂解MDC标记的α链时，其大部分荧光（约80%）定位于分子量为29,000的片段，该片段与分子量为21,000的标记纤溶酶肽具有相同的氨基末端序列。这两种肽都被Ancrod酶解成两个荧光大致相等的含受体位点的肽。这些肽的初步氨基末端序列分析与上述发现相结合，表明这两个其他的交联受体位点位于一个肽段中，该肽段占Aα链中间的17%。
