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 protein did not aggregate even when exposed to 75 degrees C for 24 h. In this study, electrostatic repulsion was shown to be responsible for the absence of aggregates at that pH. While RNase A aggregation was prevented 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 an attempt to provide the protein with excess surface cations or anions. At 75 degrees C, SDS and dextran sulfate were successful in preventing RNase A aggregation, whereas their cationic, nonionic, and zwitterionic analogs did not do so. We believe that the SO3- groups present in both additives transformed the protein into polyanionic species, and this may have provided a sufficient level of electrostatic repulsion at pH 7.8 and 75 degrees C to prevent aggregation from proceeding.
Tsai
A M
AM
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
van Zanten
J H
JH
Betenbaugh
M J
MJ
D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D055672:Static Electricity
D011506:Proteins; D012259:Ribonuclease, Pancreatic