HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti.
In investigations aimed at characterizing snake venom blood clot-dissolving enzymes, we have developed a rapid two-step high-performance chromatography method for the isolation of these fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. The first step consisted of hydrophobic interaction chromatography on a propyl-aspartamide column. Fractions containing the fibrinolytic activity were then concentrated and applied to a hydroxylapatite column. The resulting preparation, assessed for purity by reverse-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was homogeneous. The molecular weight of both venom fibrinolytic enzymes was approximately 23,000 and amino acid analysis, immunological cross-reaction, cyanogen bromide, and tryptic digestion indicate a significant degree of structural similarity. However, the general proteolytic activity of the A. p. conanti venom enzyme was significantly lower than the corresponding activity of the A. c. contortrix venom, whereas their fibrinolytic activities were quite similar.
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Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
Markland
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003435:Crotalid Venoms / Q000737:chemistry*; D004798:Enzymes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D005343:Fibrinolytic Agents / Q000302:isolation & purification*; D007120:Immunochemistry; D015394:Molecular Structure; D008970:Molecular Weight
D000596:Amino Acids; D003435:Crotalid Venoms; D004798:Enzymes; D005343:Fibrinolytic Agents