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. 2012 Mar 7;134(9):4057-9.
doi: 10.1021/ja211931f. Epub 2012 Feb 21.

A Potent, Versatile Disulfide-Reducing Agent From Aspartic Acid

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Free PMC article

A Potent, Versatile Disulfide-Reducing Agent From Aspartic Acid

John C Lukesh 3rd et al. J Am Chem Soc. .
Free PMC article

Abstract

Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pK(a) values that are ~1 unit lower than those of DTT and forms a disulfide with a similar E°' value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution.

Figures

Scheme 1
Scheme 1
Figure 1
Figure 1
Time-course for the reduction of a mixed disulfide in small molecules by DTBA and DTT in 50 mM potassium phosphate buffer. (A) Reduction of oxidized βME; kobsDTBA/kobsDTT = 3.5 at pH 7.0; kobsDTBA/kobsDTT = 4.4 at pH 5.5. (B) Reduction of oxidized l-glutathione; kobsDTBA/kobsDTT = 5.2 at pH 7.0.
Figure 2
Figure 2
Time-course for the reduction of a mixed disulfide in enzymic active sites by DTBA and DTT in 0.10 M imidazole–HCl buffer, pH 7.0, containing EDTA (2 mM). (A) Reduction of papain-Cys35–S–S–CH3; kobsDTBA/kobsDTT = 14. (B) Reduction of creatine kinase-Cys283–S–S–l-glutathione; kobsDTBA/kobsDTT = 1.1. Insets: Electrostatic potential maps with red = anionic and blue = cationic, as generated by the program PyMOL (Schrödinger, Portland, OR) using PDB entries 1ppn(19b) and 2crk.

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