Purification and Molecular Properties of Human Serum Paraoxonase-1?
H A Mehrani
1
, *
,
L Golmanesh
2
,
F Bahrami
2
and
S MTabei
3
Trauma Monthly: 12 (2) ; 139-151 Article Type: Research Article
Received:
January 1, 2007
Accepted:
January 1, 2007
To Cite:
Mehrani
H, Golmanesh
L, Bahrami
F,
S. Purification and Molecular Properties of Human Serum Paraoxonase-1?,
Trauma Mon.
Online ahead of Print
; 12(2):139-151.
Abstract
Objective. Aim of this study was to purify Human serum paraoxonase-1, which is one of the important organophosphate detoxifying enzymes.Material and Methods. In this study we found anion exchange DEAE Sephadex A-50 and gel filtration media Sephadex G-200 is suitable for purification of this enzyme. Using Triton X-100 a non ionic detergent, enzyme was separated from lipoproteins. Then enzyme solution was applied to DEAE column and washed to elute non specific binding. The bind enzyme was eluted with sodium chloride gradient. Active fractions were pooled and applied to G-200 column. The active fractions were applied to the second DEAE column. Changing the buffer and the pH resulted to a purified enzyme.Results. Results of these chromatographic procedures showed purified enzyme with greater than 95% purity and 320 U/L of specific activity. SDS –Poly acryl amid gel electrophoresis showed a single band of approximately 43 KD protein. Kinetic properties of the purified enzyme showed that, affinity of the enzyme to the substrates is dependent on buffer composition and the calcium and sodium ions concentration. Using paraoxon as the substrate the activity was related to the calcium and sodium concentration (Km = 1.39 ± 0.52), but for phenyl acetate as the substrate the activity was independent of sodium and calcium (Km = 0.728 ± 0.105). The optimal pH for paraoxon hydrolysis was around 9.5-11, but for phenyl acetate it was from 8-8.5. Glycerol 20% (v/v) was most effective stabilizer. Keeping at 25 °C for 20 days 75% of the original activity was restored. Conversely at 56 °C the ammonium sulfate was better stabilizer. EDTA and zinc chloride both inhibited the enzyme activity. Concomitant incubation of the inhibitors with calcium ion resulted to increased IC50 values.Conclusion. The stablished purification procedure is simple and non-expensive and can be used for large scale purification of this enzyme which can be used for organophosphate decontamination or prevention of intoxication.
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