1Thomas J. Long School of Pharmacy, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211
2Section of Microbiology, University of California, Davis, CA 95616
3 Current affiliation: CovX, 9381 Judicial Dr., Suite 200, San Diego, CA 92121
*For Correspendence: xli@pacific.edu
The effect of experimental conditions on the formation of structural intermediates and pathway of bovine insulin fibril formation was studied at pH 0.5 and 60°C. The relative amount of oligomeric intermediates observed during fibril formation was found to depend on both ionic strength and protein concentration. At a protein concentration of 1mg/mL, significant amounts of oligomeric intermediates including spherical assemblies and protofibrils were detected by AFM under the condition of 0.35 ionic strength. The oligomeric intermediates dissociated upon cooling for 10 days at 25°C. At 0.15 ionic strength, fibril formation proceeded without generation of oligomeric intermediates. Amorphous aggregates were observed prior to detection of well-defined fibrils under this condition. Increasing protein concentration from 1 to 2 mg/mL at 0.15 ionic strength or decreasing the protein concentration from 1 to 0.5 mg/mL at 0.35 ionic strength resulted in generation of similar low amounts of oligomeric intermediates. Fibril formation at 0.35 ionic strength and 1mg/mL of insulin was consistent with a nucleated conformational conversion mechanism, while fibril formation at 0.15 ionic strength and at the same protein concentration followed a nucleated polymerization mechanism. The results from this study show that ionic strength and protein concentration determined the relative quantities of structural intermediates formed during the early stages of fibril formation and the pathway of fibril assembly.
Bovine insulin, fibril, protofibril, AFM
