ISSN:
0006-3592
Keywords:
heat denaturation
;
protein aggregation
;
RNase A
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, Tm, of approximately 65°C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around Tm using near- and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements. These measurements revealed the presence of folded structures at 55°C and below, while denatured structures appeared at 65°C and above. Concurrent static light scattering (SLS) measurements, employed to detect the presence of RNase A aggregates, showed that RNase A aggregation was observed at 65°C and above, when much of the protein was denatured. Subsequent NMR time-course data demonstrated that aggregates forming at 75°C and pH 7.8 were indeed derived from heat-denatured protein. However, aggregation was also detected at 55°C when the spectroscopic data suggested the protein was present predominantly in the folded configuration. In contrast, heat denaturation did not lead to RNase A aggregation in a very acidic environment. We attribute this phenomenon to the effect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:273-280, 1998.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
