Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts

Abstract

Artifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction−oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds.