Arginine and the Hofmeister Series: the role of ion-ion interactions in protein aggregation suppression.

L-Arginine hydrochloride is a very important aggregation suppressor for which there has been much attention given regarding elucidating its mechanism of action. Little consideration, however, has been given toward other salt forms besides chloride, even though the counterion likely imparts a large influence per the Hofmeister Series. Here, we report an in depth analysis of the role the counterion plays in the aggregation suppression behavior of arginine. Consistent with the empirical Hofmeister series, we found that the aggregation suppression ability of other arginine salt forms on a model protein (α-chymotrypsinogen) follows the order: H(2)PO(4)(-) > SO(4)(2-) > citrate(2-) > acetate(-) ≈ F(-) ≈ Cl(-) > Br(-) > I(-) ≈ SCN(-). Mechanistically, preferential interaction and osmotic virial coefficient measurements, in addition to molecular dynamics simulations, indicate that attractive ion-ion interactions, particularly attractive interactions between arginine molecules, play a dominate role in the observed behavior. Furthermore, it appears that dihydrogen phosphate, sulfate, and citrate have strong attractive interactions with the guanidinium group of arginine, which seems to contribute to the superior aggregation suppression ability of those salt forms by bridging together multiple arginine molecules into clusters. These results not only further our understanding of how arginine influences protein stability, they also help to elucidate the mechanism behind the Hofmeister Series. This should help to improve biopharmaceutical stabilization through the use of other arginine salts and possibly, the development of novel excipients.