Abstract

Human paraoxonases-1 is one of the most important detoxifying enzymes. In this study using simple chromatographic procedures human paraoxonases-1 was purified from human pooled plasma. The enzyme was purified using DEAE Sephadex an anion exchanger and G-200 a gel filtration chromatographic media. Results showed a single band of approximately 43 KID proteins in SIDS-PAGE, corresponding to the human PON1. Using paraoxon as the substrate the activity was related to the concentration of calcium and sodium ions (K-m = 1.2 +/- 0.2 mM). Phenyl acetate hydrolyzing activity was independent of sodium and calcium ions (K-m=0.78 +/- 0.08 mM). Keeping at 25 degrees C for 20 days 75 of the enzyme original activity was restored in 20 (v/v) glycerol. EDTA and zinc chloride both inhibited the enzyme activity. In conclusion the applied procedures can be used for large scale purification. It would greatly facilitate their structural and functional characterization and permit examination of their weak, yet potentially most biologically relevant activities, in the complete absence of other serum proteins. (c) 2007 Elsevier B.V. All rights reserved.