Further characterization of the anticoagulant proteinase, cerastase F-4 from Cerastes cerastes (Egyptian sand viper) venom.
A potent anticoagulant, cerastase F-4, was purified from the venom of Cerastes cerastes. The u.v. absorption spectrum revealed a relatively high tyrosine and low tryptophan content. The molar extension coefficient and E278(0.1%) were 19,400 and 0.84, respectively. The enzyme secondary structure, as studied by circular dichroism, showed 23.6% alpha-helix, 34% beta-sheets, 19% beta-turns and 32.5% random coils. When casein was used as a substrate the optimum pH was 10.0 and the Km was 1.45 g/l. Cerastase F-4 is a metallo-enzyme that contains one mole of Ca2+ and one mole of Zn2+ per mole of protein. It is not affected by phenylmethane sulfonylfluoride or soybean trypsin inhibitor, while it is completely inhibited by 0.5 mM EDTA or ethyleneglycol bis (beta-amino ethylether) N,N,N',N'-tetraacetic acid (EGTA). Ca2+, Mg2+ and Zn2+ partially activated the enzyme under different experimental conditions. Our results suggest that Ca2+ and Zn2+ may play a role in maintaining the structural and catalytic integrity of the enzyme.
D000818:Animals; D000925:Anticoagulants / Q000032:analysis*; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000032:analysis*; D005340:Fibrinogen / Q000494:pharmacology; D005343:Fibrinolytic Agents; D006801:Humans; D066298:In Vitro Techniques; D008670:Metals / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D014757:Viper Venoms / Q000032:analysis*
D000925:Anticoagulants; D002614:Chelating Agents; D005343:Fibrinolytic Agents; D008670:Metals; D011480:Protease Inhibitors; D014757:Viper Venoms; D005340:Fibrinogen; D004492:Edetic Acid; D010450:Endopeptidases; C048743:cerastase F-4