Separation of Crotalus atrox (western diamondback rattlesnake) alpha-proteinase from serine proteinase and hemorrhagic factor activities.
A method for obtaining Crotalus atrox alpha-proteinase (EC 3.4.24.1) in a pure form has been developed. Fractionation of the crude venom on DEAE-Sepharose, followed by gel filtration on Bio-Gel P-150 and chromatography on CM-Sepharose, yielded an alpha-proteinase preparation which showed a single band on disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had an activity on casein approximately twice that previously reported. The enzyme is a nonglycosylated single-chain polypeptide with a molecular weight of 26,738 and a pI of 8.15. Proteolytic activity on casein, alpha 1-antichymotrypsin, and Cl-inhibitor was abolished by treatment of alpha-proteinase with 1 mM EDTA, but full activity was retained in the presence of 1 mM phenylmethylsulfonyl fluoride. Caseinolytic activity was increased by 33 and 55% in the presence of 10 mM Mg2+ and Ca2+, respectively. Pure alpha-proteinase is devoid of esterolytic activity on H-D-Pro-Phe-Arg-p-nitroanilide (S-2302), benzoyl-L-arginine ethyl ester, and benzoyl-L-tyrosine ethyl ester. The final preparation has no hemorrhagic factor activity.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003435:Crotalid Venoms / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010450:Endopeptidases / Q000302:isolation & purification*; D008666:Metalloendopeptidases; D008667:Metalloproteins / Q000302:isolation & purification*; D008970:Molecular Weight; D012697:Serine Endopeptidases
D000596:Amino Acids; D003435:Crotalid Venoms; D008667:Metalloproteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; C044577:atrolysin A; C041374:atrolysin F