Paper Information

Title:  PURIFICATION, STABILIZATION AND MOLECULAR PROPERTIES OF HUMAN SERUM PARAOXONASE-1
Type: POSTER
Author(s): HOSSEINI MEHRAN,GOLMANESH L.,TABEI S.M.
 
 
 
Name of Seminar: IRANIAN CONGRESS OF PHYSIOLOGY AND PHARMACOLOGY
Type of Seminar:  CONGRESS
Sponsor:  PHYSIOLOGY AND PHARMACOLOGY SOCIETY, MASHHAD UNIVERSITY OF MEDICAL SCIENCE
Date:  2007Volume 18
 
 
Abstract: 

Organophosphates and carbamates compounds are used in pest control in agriculture and as insecticides in household conditions. Aannually more than 3 million poisoning and 200,000 deaths occur with this intoxication. Human paraoxonases-1 which is attached to high density lipoproteins (HDL) seems to be one of the important detoxifying enzymes. In this study using different analytical and laboratory methods human paraoxonases-1 was purified from human outdated sera. Trial and errors showed that the anion exchange DEAE Sephadex A-50 and gel filtration media Sephadex G-200 is suitable for purification. 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 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 proteins. 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. Using this purification procedure large amount of purified enzyme can be used for organophosphate decontamination or prevention of intoxication.

 
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