Studies on chemically aggregated pepsin using glutaraldehyde.
Porcine pepsin was immobilized by chemical aggregation using glutaraldehyde as a bifunctional crosslinking agent. The immobilized pepsin followed Michaelis-Menten kinetics (K(m) = 5.3 x 10(-5) M) and the yield of immobilization was 91%. The activation energy of the immobilized preparation was 90,613 cal/mol as compared to 67,532 cal/mol for native pepsin. Using acid-denatured hemoglobin and N-acetyl phenyl-alanyl-3, 5-diiodotyrosine (APDT) as substrates, the activities shown by the immobilized pepsin were, respectively, 67 and 79% that of the soluble pepsin. The immobilized pepsin showed marked stabilization against pH, temperature, urea, and guanidine hydrochloride. The activity of the immobilized preparation in the presence of urea was greater when hemoglobin was used as the substrate than when APDT was used as substrate. Storage of the preparation under refrigerated conditions for 160 days showed 58% retention in enzyme activity. The immobilized pepsin can be removed from the reaction mixture volume easily, retaining nearly 100% of its activity even after being used in seven consecutive assays.
Biochemistry Division, Department of Chemistry, Aligarh Muslim University, Aligarh 202001, UP, India.
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Siddiqi