The purification of the enzyme hydrolysing diethyl p-nitrophenyl phosphate (paraoxon) in sheep serum

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Abstract
The enzyme in sheep serum which hydrolyzes diethyl p-nitrophenyl phosphate (paraoxon) has been purified 330- to 385-fold. Electrophoretic evidence suggests that the product was 80-95% pure and contained only one other, non-active, component. Low-temperature ethanol, pH and ionic-strength fractionation resulted in a 114-fold purification with a 32% yield. (NH4)2SO4 fractionation increased the purification to 300-fold and lowered the yield to about 15%. Repeated (NH4)2SO4 fractionation brought the purification to 330- to 385-fold. The paraoxon Km of purified fractions (4.2 m[image]) was 15 times that of sheep serum (0.29 m[image]). The characteristics of the increase in Km suggests the presence of a paraoxonase activator in sheep serum which was removed during purification. Paraoxonase solutions fluoresced blue when exposed to UV radiation. Analysis of the enzyme indicated the presence of about 1.5% of P and chromato-graphic experiments indicated the presence of choline-containing phospholipids. The sedimentation constant and other observations suggest a molecular weight of 35,000-50,000.