II - ELECTROSTATIC EFFECT IN THE AGGREGATION OF HEAT-DENATURED RNASE-A AND IMPLICATIONS FOR PROTEIN ADDITIVE DESIGN

In the previous study (part I), heat-denatured RNase A aggregation was shown to depend on the solution pH. Interestingly, at pH 3.0, the pro tein did not aggregate even when exposed to 75 degrees C for 24 h. In this study, electrostatic repulsion was shown to be responsible for th e absence of aggregates at that pH. While RNase A aggregation was prev ented at the extremely acidic pH, this is not an environment conducive to maintaining protein function in general. Therefore, attempts were made to confer electrostatic repulsion near neutral pH. In this study, heat-denatured RNase A was mixed with charged polymers at pH 7.8 in a n attempt to provide the protein with excess surface cations or anions . At 75 degrees C, SDS and dextran sulfate were successful in pre vent ing RNase A aggregation, whereas their cationic, nonionic, and zwitter ionic analogs did not do so. We believe that the SO, groups present in both additives transformed the protein into polyanionic species, and this may have provided a sufficient level of electrostatic repulsion a t pH 7.8 and 75 degrees C to prevent aggregation from proceeding. (C) 1998 John Wiley & Sons, Inc.