Detection of heparin-like glycosaminoglycans in normal human plasma by polyacrylamide-gel electrophoresis.
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.
Cavari
S
S
Institute of General Pathology, University of Florence, Firenze, Italy.
Vannucchi
S
S
D004588:Electrophoresis, Cellulose Acetate; D004591:Electrophoresis, Polyacrylamide Gel; D006025:Glycosaminoglycans / Q000097:blood*; D006493:Heparin / Q000097:blood*; D006801:Humans; D006868:Hydrolysis; D012016:Reference Values
D006025:Glycosaminoglycans; D006493:Heparin