Mojave rattlesnake ( Crotalus scutulatus scutulatus) venom: Enzyme activities and purification of arginine ester hydrolases

Abstract

Venom from the Mojave rattlesnake was fractionated on DEAE Sephadex into 12 fractions (MD-1–12). Each fraction was assayed for five enzymatic activities, all known to occur in crotalid venoms. Phosphomonoesterase activity was not present in either the crude venom or any of the fractions. l-Amino acid oxidase activity was found in several fractions (MD-4–9), being eluted from the column as a broad peak of activity. Phosphodiesterase activity was found in MD-1–2, eluting from the column as a single peak of activity. Phospholipase activity was fractionated into three peaks of activity in MD-2–4, MD-7 and MD-9. The phospholipase activity in MD-9 was associated with Mojave toxin. Arginine ester hydrolase activity was distributed as several peaks of activity throughout MD-1–9. Two arginine ester hydrolases (AAEI, AAEII) were isolated from Mojave venom by fractionation on DEAE Sephadex followed by chromatofocusing chromatography and affinity chromatography. They were purified to specific activities of 60 U/mg (AAEI) and 36.1 U/mg (AAEII) with BAEE as substrate at pH 7.5. This procedure showed per cent yields of 5.0% for AAEI and 1.0% for AAEII. The two proteins were homogeneous by PAGE, narrow range isoelectric focusing and SDS gel electrophoresis. Both proteins were acidic, with pI values equal to 4.7 (AAEI) and 4.4 (AAEII). The molecular weights as determined by SDS gel electrophoresis were 33,200 for AAEI and 34,700 for AAEII.