Purification and anticoagulant activity of glycosaminoglycans isolated from the skin of common carp, Cyprinus carpio

Abstract

Biochemical investigations are important tools for discovery of new anticoagulant agents present in a variety of aquatic organisms to substitute for the traditional use of heparin (HEP) in the medical clinic. The aim of this study was to purify and evaluate the anticoagulant activity of glycosaminoglycans (GAGs) isolated from the skin of the common carp Cyprinus carpio. GAGs were extracted with crude papain in 0.1 sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by ion exchange chromatography on DEAE-cellulose column where five fractions were eluted (F I; F II; F III; F IV e F V) using a NaCl gradient. The fractions were concentrated by lyophilization and submitted to 0.5% agarose gel electrophoresis. The anticoagulant activity was evaluated by the activated partial thromboplastin time (APTT) using normal human plasma and expressed in international units per mg of polysaccharide using standard HEP (193 IU mg^-1). The methodology was efficient in the extraction and fractionation of GAGs, and the preliminary identification by electrophoresis suggested molecules of dermatan sulfate and preponderance of heparan sulfate. The anticoagulant activity expressed as dose-dependence manner, however resulting in a maximum of 4.89 IU mg^-1 compared to heparin. Therefore, the use of specific degradation enzymes to characterization of heparinoid obtained from the proteolytic digestion in isolation of GAGs presented in the skin of C. carpio suggests posterior studies. The employment of technique with other cultivated species will be also indicated.