I - STUDY OF PROTEIN AGGREGATION DUE TO HEAT DENATURATION - A STRUCTURAL APPROACH USING CIRCULAR-DICHROISM SPECTROSCOPY, NUCLEAR-MAGNETIC-RESONANCE, AND STATIC LIGHT-SCATTERING

The objective of this study was to investigate the relationship betwee n oxidized RNase A protein structure and the occurrence of protein agg regation using several spectroscopic techniques. Circular dichroism sp ectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, T-m, of approxim ately 65 degrees C in deionized water. A more detailed era mi nation o f the protein structure was undertaken at several temperatures around T-m using near- and far-UV CD and one-dimensional nuclear magnetic res onance (NMR) measurements. These measurements revealed the presence of folded structures at 55 degrees C and below, while denatured structur es appeared at 65 degrees C and above. Concurrent static light scatter ing (SLS) measurements, employed to detect the presence of RNase A agg regates, showed that RNase A aggregation was observed at 65 degrees C and above, when much of the protein was denatured. Subsequent NMR time -course data demonstrated that aggregates forming at 75 degrees C and pH 7.8 were indeed derived from heat-denatured protein. However, aggre gation was also detected at 55 degrees C when the spectroscopic data s uggested the protein was present predominantly in the folded configura tion. In contrast, heat denaturation did not lead to RNase A aggregati on in a very acidic environment. We attribute this phenomenon to the e ffect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH. (C) 1998 John Wiley & Sons, Inc.