Partial purification and kinetic studies of Eosinophil peroxidase enzyme from hemolysate blood cells in mice induced atherosclerosis and inhibition effects of some isoflavone compounds

Nahida Saieed Al-Chalabi, Eman Saieed Al-Choka

Abstract


This study include an isolation, partial purification and study of the  kinetic properties of Eosinophil peroxidase (EPO) enzyme from hemolysate blood cells in induced atherosclerosis and normal mice, then comparison between them.

Isolation of two protein peaks I and II were carried out using ion exchange chromatography (DEAE cellulose), specific activity for these two peaks I and II for EPO enzyme for normal and induced atherosclerosis were (0.018, 0.02) and (0.126, 0.031). unit mg -1 of protein respectively, peak I has a high specific activity for EPO enzyme which was isolated from induced atherosclerosis mice.

The optimum condition of EPO for the peak I separated from hemolysate blood cells in induced atherosclerosis mice showed an optimum reaction incubation time at 21 minutes, pH of  sodium acetate buffer 6, temperature at 30 °C, volume of enzyme 500 μL and the substrate concentration was about, 200 μM.

When we used Line Weaver-Burk plot, the maximum velocity (Vmax) and Michale’s - Menten constant (Km) were found to be 0.045 unit ml-1 and 400 μM L-1 respectively, all cationic metal ions such as Mg+2, Na+, Ca+2, K+, Mn+2, Hg+2 (10 mM) shows a catalytic effect on the activity of EPO enzyme but maximum that for Hg+2 ions.

Also at this study shows an inhibition effect of some isoflavone compounds such as genistein, daidzein, biochanin A and  formononetin on the activity of this enzyme. The results indicate different relative inhibition in enzyme activity, for these compounds, according to Line Weaver- Burk plot genistein compound shows a competitive inhibition whereas daidzein, biochanin A and formononetin shows  non competitive inhibition for EPO enzyme.

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DOI: http://dx.doi.org/10.17628/ecb.2014.3.961-968

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