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Acidic Dithiothreitol Reduction on Disulfide Bonds using Cation‐Exchange Chromatography

Abstract Dithiothreitol has been utilized to reduce the disulfide bonds between homocysteine and other thiols in total plasma homocysteine determination. The completeness of the reduction of disulfides into thiols is a prerequisite for accurate quantitation of total plasma homocysteine. In order to evaluate the effectiveness of dithiothreitol reduction on disulfide bonds, the acidic mixture of cystine and homocystine is reduced under controlled parameters including dithiothreitol concentration, reduction temperature, and reaction time. The reaction mixture is analyzed by cation‐exchange chromatography. It is concluded that 1.0 M dithiothreitol concentration, 70°C reduction temperature, and 1 hour reaction time are required to convert more than 96% of the disulfides of cystine and homocystine to their thiols in acidic medium. This study also proves that dithiothreitol is able to keep cysteine and homocysteine from being reoxidized back to their disulfides.