Analysis of protein back-extraction processes in alcohol- and carboxylic acid-mediated AOT reverse micellar systems based on structural changes of proteins and reverse micelles

Abstract

The effects of the addition of alcohol and carboxylic acid on the back-extraction of several small globular proteins such as β-lactoglobulin (β-LG), bovine carbonic anhydrase (CAB), and lipase have been studied using reverse micellar systems (RVMS). The protein back-extraction induced by the addition of alcohol and carboxylic acid was markedly influenced by the structural changes of reverse micelles ( β t) and protein ( m). The alcohols and carboxylic acids suppressing the formation of reverse micelle clusters (positive value of β t) remarkably enhanced the back-extraction of proteins. On the other hand, the alcohols and carboxylic acids enhancing the formation of reverse micelle clusters (negative value of β t) suppressed the back-extraction of proteins. The markedly effective alcohol and carboxylic acid molecules on the denaturation of proteins (high values of m) suppressed the back-extraction of proteins. The m values were estimated by measuring the dependence of the free energy change of denaturation on the concentration of alcohol or carboxylic acid, and were correlated well with the solvent-accessible surface area (ASA) of alcohol and carboxylic acid molecules. We have constructed a simple equation on the basis of these factors for explaining the effect of alcohols and carboxylic acids in back-extraction processes of proteins of RVMS. These results suggested that the back-extraction processes were controlled by the formation of reverse micelle clusters as well as the denaturation of proteins.