Isolation and Studying Cathepsin B Enzyme from Normal Human Serum

R. Saduon, Rafad; Y. Ahmad, Tareq

doi:10.33899/rjs.2011.6519

	Isolation and Studying Cathepsin B Enzyme from Normal Human Serum
Rafidain Journal of Science
Article 7, Volume 22, Issue 6, 2011, Pages 73-87 PDF (0 K)
DOI: 10.33899/rjs.2011.6519
Authors
Rafad R. Saduon; Tareq Y. Ahmad
Abstract
The work included the isolation of cathepsin B enzyme from normal human serum using different biochemical techniques which included: ammonium sulfate precipitation, dialysis and gel filtration chromatography on sephadex G-100. Furthermore, the comparative molecular weight of cathepsin B enzyme which is partially isolated from serum was found to be (24,000 ± 500 Da) by gel filtration chromatography. The results also showed that the optimum conditions of cathepsin B enzyme activity were obtained using sodium phosphate (100 mmol/L) as a buffer at pH (6.0), at a temperature (40 ˚C) and (1 mmol/L) of BANA as a substrate. Using Linweaver Burk plot, it was found that Vmax and Km have the values of (346.20 U/L) and (0.489 mmol/L) respectively. The results also showed that the activity of cathepsin B enzyme was increased using thiol compound. Cysteine showed a greater activation than β-mercaptoethanol. The activation of cathepsin B enzyme was greater in the presence of EDTA in the reaction mixture. Finally, the effect of iodoacetate, calcium chloride, mercury chloride, zinc sulfate and potassium cyanide on the activity of cathepsin B was studied. These compounds showed strong inhibition on the activity of cathepsin B. The inhibition of cathepsin B by mercury chloride was irreversible type of inhibition at a concentration (0.01 mmol/L).
Keywords
Cathepsin B; isolation; human serum; BANA; inhibition

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