PURIFICATION AND MOLECULAR PROPERTIES OF HUMAN SERUM PARAOXONASE-1

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MEHRANI H.A.; GOLMANESH L.; BAHRAMI F.; TABEI M.

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Journal: KOWSAR MEDICAL JOURNAL Year:2007 | Volume:12 | Issue:2 Page(s): 139-151

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Title

PURIFICATION AND MOLECULAR PROPERTIES OF HUMAN SERUM PARAOXONASE-1

Author(s)

MEHRANI H.A. | GOLMANESH L. | BAHRAMI F. | TABEI M. | Issue Writer Certificate

Keywords

PARAOXONASE-1Q3

PROTEIN PURIFICATIONQ3

PARAOXONQ4

PHENYL ACETATEQ3

Abstract

Abstract‎ Objective: Aim of this study was to purify Human serum PARAOXONASE-1, which is one of the ‎important organophosphate detoxifying enzymes. Materials 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 25oC for 20 days 75% of the original activity was ‎restored. Conversely at 56oC 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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APA: Copy

MEHRANI, H.A., GOLMANESH, L., BAHRAMI, F., & TABEI, M.. (2007). PURIFICATION AND MOLECULAR PROPERTIES OF HUMAN SERUM PARAOXONASE-1. KOWSAR MEDICAL JOURNAL, 12(2), 139-151. SID. https://sid.ir/paper/32823/en

Vancouver: Copy

MEHRANI H.A., GOLMANESH L., BAHRAMI F., TABEI M.. PURIFICATION AND MOLECULAR PROPERTIES OF HUMAN SERUM PARAOXONASE-1. KOWSAR MEDICAL JOURNAL[Internet]. 2007;12(2):139-151. Available from: https://sid.ir/paper/32823/en

IEEE: Copy

H.A. MEHRANI, L. GOLMANESH, F. BAHRAMI, and M. TABEI, “PURIFICATION AND MOLECULAR PROPERTIES OF HUMAN SERUM PARAOXONASE-1,” KOWSAR MEDICAL JOURNAL, vol. 12, no. 2, pp. 139–151, 2007, [Online]. Available: https://sid.ir/paper/32823/en

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