Differential hydrolysis of homocysteine thiolactone by purified human serum (192)Q and R-192 PON1 isoenzymes


Bayrak A., Bayrak T., Demirpence E., Kilinc K.

JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, vol.879, no.1, pp.49-55, 2011 (Peer-Reviewed Journal) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 879 Issue: 1
  • Publication Date: 2011
  • Doi Number: 10.1016/j.jchromb.2010.11.006
  • Journal Name: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
  • Journal Indexes: Science Citation Index Expanded, Scopus
  • Page Numbers: pp.49-55

Abstract

Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human (192)Q and R-192 PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for R-192 PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45 kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K-m values of (192)Q and R-192 PON1 for homocysteine thiolactone were 23.5 mM and 22.6 mM respectively. For R-192 PON1, the V-max was 2.5-fold and k(cat)/K-m was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and R-192 PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment. (C) 2010 Elsevier B.V. All rights reserved.