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.

[1]  G. Boers,et al.  Improved identification of heterozygotes for homocystinuria due to cystathionine synthase deficiency by the combination of methionine loading and enzyme determination in cultured fibroblasts , 2004, Human Genetics.

[2]  S. Sengupta,et al.  A facile synthesis of homocysteine-cysteine mixed disulfide. , 2001, Analytical biochemistry.

[3]  Michael Williams,et al.  TOTAL PLASMA HOMOCYSTEINE DETERMINATION USING ION-EXCHANGE CHROMATOGRAPHY , 2000 .

[4]  D. Jacobsen Acquired hyperhomocysteinemia in heart transplant recipients. , 1998, Clinical chemistry.

[5]  D. Jacobsen Homocysteine and vitamins in cardiovascular disease. , 1998, Clinical chemistry.

[6]  D. A. Cramer Homocysteine vs Cholesterol: Competing Views, or a Unifying Explanation of Arteriosclerotic Cardiovascular Disease? , 1998 .

[7]  M. Tsai Laboratory assessment of mild hyperhomocysteinemia as an independent risk factor for occlusive vascular diseases. , 1996, Clinical chemistry.

[8]  B. Hultberg,et al.  Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization. , 1993, Clinical chemistry.

[9]  P. Ueland,et al.  Fully automated fluorescence assay for determining total homocysteine in plasma. , 1989, Clinical chemistry.

[10]  D. Rabenstein,et al.  Determination of homocysteine, penicillamine, and their symmetrical and mixed disulfides by liquid chromatography with electrochemical detection. , 1989, Analytical biochemistry.

[11]  R. Green,et al.  Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection. , 1989, Analytical biochemistry.

[12]  W. Baeyens,et al.  Hplc Determination of N-Acetylcysteine in Pharmaceutical Preparations After Pre-Column Derivatization With ThiolyteR MB Using Fluorescence Detection , 1988 .

[13]  W. Baeyens,et al.  Effects of reducing agents on the determination of thiolic compounds in the presence of their disulfides using bimane pre-column derivatization , 1987 .

[14]  W. Cleland DITHIOTHREITOL, A NEW PROTECTIVE REAGENT FOR SH GROUPS. , 1964, Biochemistry.