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Date of Award

2005

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Xiaoling Li

Second Advisor

Bhaskara Jasti

First Committee Member

David Thomas

Second Committee Member

James Uchizono

Third Committee Member

Wei Wang

Abstract

The effect of environmental condition on the mechanism and kinetics of fibril formation for bovine insulin were investigated. Results showed environmental conditions played a significant role in determining the mechanism and kinetics of fibril formation. Increased protein concentration, elevated temperature, and higher ionic strengths induced insulin to form fibrils through oligomeric intermediates that were consistent with the nucleated conformational conversion (NCC) mechanism. Bovine insulin was also shown to generate fibrils without formation of oligomeric intermediates at study conditions of lower protein concentration, lower temperature, and lower ionic strength. Fibril formation without oligomeric intermediate can be described by the nucleated polymerization (NP) mechanism. Different relative amounts of oligomeric intermediate were generated at the various combinations of protein concentration, temperature, and ionic strength. The kinetic parameters, lag time, and rate of fibril formation, correlated with the relative amount of oligomeric intermediates detected. Longer lag times and slower rates of fibril formation were observed with greater amounts of oligomeric intermediate present. The effects of excipients, trifluoroethanol, ethanol, glycerol, and urea on the apparent rate constants of oligomeric intermediate and fibril formation were also investigated. At the concentrations studied, all the excipients tested were observed to decrease the rate and relative amount of oligomeric intermediate formation in an excipient concentration-dependent manner. The excipients were less effective at preventing fibril formation. In conclusion, bovine insulin can form fibrils with and without oligomeric intermediates. Protein concentration and environmental conditions, such as temperature, ionic strength, and excipients played a significant role in determining the relative amount of oligomeric intermediates, which in turn, determined the mechanism and kinetics of bovine insulin fibril formation under the conditions studied.

Pages

189

ISBN

9780542028847 , 0542028840

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