Cavari S, Vannucchi S
Institute of General Pathology, University of Florence, Firenze, Italy.
Clin Chim Acta. 1996 Aug 30;252(2):159-70. doi: 10.1016/0009-8981(96)06331-0.
This paper describes a procedure that allows detection of endogenous heparin present in normal human plasma by polyacrylamide-gel electrophoresis (PAGE). Plasma was submitted to proteolysis: an antithrombin III-dependent and heparinase I-sensitive anticoagulant activity was demonstrated in the supernatant of the digest. The supernatant was submitted to sequential fractionation with increasing concentrations of ethanol (25%, 50%, 60% and 65%, by vol.). Fractions were analyzed by PAGE for both glycosaminoglycan (GAG) and protein content. GAGs were detected by gradient PAGE (24-30%). The fraction obtained by 60% ethanol precipitation contained heparinase I-sensitive GAG. We show that GAGs co-precipitate with proteins. The SDS-PAGE of the material resulting from proteolytic digestion and subsequent ethanol fractionation, revealed three major bands. These peptides co-precipitated with plasma GAGs, mainly with the fraction obtained by 60% ethanol. We discuss the possibility that circulating endogenous heparin interacts with such peptides.
本文描述了一种通过聚丙烯酰胺凝胶电泳（PAGE）检测正常人血浆中内源性肝素的方法。将血浆进行蛋白水解：在消化上清液中证明了一种抗凝血酶III依赖性且对肝素酶I敏感的抗凝活性。将上清液用浓度递增的乙醇（体积分数分别为25%、50%、60%和65%）进行连续分级分离。通过PAGE分析各馏分的糖胺聚糖（GAG）和蛋白质含量。通过梯度PAGE（24 - 30%）检测GAG。通过60%乙醇沉淀获得的馏分含有对肝素酶I敏感的GAG。我们表明GAG与蛋白质共沉淀。蛋白水解消化及随后乙醇分级分离得到的物质的SDS - PAGE显示出三条主要条带。这些肽与血浆GAG共沉淀，主要是与通过60%乙醇获得的馏分。我们讨论了循环内源性肝素与此类肽相互作用的可能性。
