Insulin aggregation in aqueous media and its effect on alpha-chymotrypsin-mediated proteolytic degradation.
Self-association of zinc-insulin monomers into dimers and hexamers may lead to enhanced protection of the peptide from proteolytic degradation. The present study has been undertaken to investigate the relationship, if any, between the rate of enzymatic degradation of insulin by a protease, alpha-chymotrypsin, and the extent of insulin aggregation in aqueous solutions. Insulin solutions (0.6 mg/ml) containing varying proportions of dimer and hexamer were obtained by adding ethylene diamine tetraacetic acid (EDTA) within a concentration range of 0.005 to 0.040 mM. As the EDTA concentration was increased above 0.040 mM, a complete dissociation of hexamers to dimers occurred and the rate of enzymatic degradation reached its maximum. The overall first-order rate constants appeared to be linearly related to the square of EDTA concentrations. The apparent first-order rate constants for dimer and hexamer degradation obtained from a linear plot of rate constant versus EDTA squared concentration were found to be 0.02800 +/- 0.00065 and 0.00798 +/- 0.00075 min-1, respectively. Two major insulin degradation products were also detected and the kinetics of product appearance agreed well with the disappearance kinetics of insulin. The results indicated that the degradation of insulin dimers by alpha-chymotrypsin is about 3.5 times faster than the degradation of the hexamer. The second-order dependency of degradation rate on EDTA concentration might be due to the fact that insulin hexamers contain two zinc ions which are sequestered by two EDTA molecules. Chelation of zinc ions by EDTA lead to hexamer deaggregation to dimers as was evidenced from a circular dichroism study.(ABSTRACT TRUNCATED AT 250 WORDS)
Liu
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Department of Industrial and Physical Pharmacy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907.
Kildsig
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Mitra
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D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000737:chemistry*; D002942:Circular Dichroism; D004492:Edetic Acid; D006868:Hydrolysis; D007328:Insulin / Q000737:chemistry*; D013552:Swine; D014867:Water
D007328:Insulin; D014867:Water; D004492:Edetic Acid; D002918:Chymotrypsin