A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry

doi:10.1006/abio.1999.4203

Analytical Biochemistry

Volume 273, Issue 1, 15 August 1999, Pages 73-80

https://doi.org/10.1006/abio.1999.4203 Get rights and content

Abstract

The newly introduced sulfhydryl reductant tris(2-carboxyethyl)phosphine (TCEP) is a potentially attractive alternative to commonly used dithiothreitol (DTT). We compare properties of DTT and TCEP important in protein biochemistry, using the motor enzyme myosin as an example protein. The reductants equally preserve myosin's enzymatic activity, which is sensitive to sulfhydryl oxidation. When labeling with extrinsic probes, DTT inhibits maleimide attachment to myosin and must be removed before labeling. In contrast, maleimide attachment to myosin was achieved in the presence of TCEP, although with less efficiency than no reductant. Surprisingly, iodoacetamide attachment to myosin was nearly unaffected by either reductant at low (0.1 mM) concentrations. In electron paramagnetic resonance (EPR) spectroscopy utilizing nitroxide spin labels, TCEP is highly advantageous: spin labels are two to four times more stable in TCEP than DTT, thereby alleviating a long-standing problem in EPR. During protein purification, Ni²⁺ concentrations contaminating proteins eluted from Ni²⁺ affinity columns cause rapid oxidation of DTT without affecting TCEP. For long-term storage of proteins, TCEP is significantly more stable than DTT without metal chelates such as EGTA in the buffer, whereas DTT is more stable if metal chelates are present. Thus TCEP has advantages over DTT, although the choice of reductant is application specific.

References (28)

P.C. Jocelyn
Methods Enzymol.
(1987)
L.E.S. Netto et al.
Arch. Biochem. Biophys.
(1996)
D.O. Lambeth et al.
Biochim. Biophys. Acta
(1982)
J.C. Han et al.
Anal. Biochem.
(1994)
A.G. Weeds et al.
J. Mol. Biol.
(1977)
J.A. Spudich et al.
J. Biol. Chem.
(1971)
R. Takashi et al.
Arch. Biochem. Biophys.
(1976)
J.C. Crawhall et al.
Biochim. Biophys. Acta
(1966)
E. Graf et al.
J. Biol. Chem.
(1984)
W.W. Cleland
Biochemistry
(1964)

J.A. Burns et al.

J. Org. Chem.

(1991)

J. Podlaha et al.

Collect. Czech. Chem. Commun.

(1973)

U.T. Ruegg et al.

Methods Enzymol.

(1977)

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^☆: This work was supported by NIH Grants HL32145 (R.C.) and AR44420 (P.R.S.), and by Bank of America–Giannini Foundation and UC Presidents Postdoctoral Fellowships (E.B.G.).

²: To whom correspondence should be addressed at Loomis Laboratory of Physics, 1110 W. Green St., University of Illinois, Urbana IL 61801. Fax: (217) 244-7187. E-mail: [email protected].

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Regular ArticleA Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry☆

Abstract

Methods Enzymol.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

Anal. Biochem.

J. Mol. Biol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochemistry

J. Org. Chem.

Collect. Czech. Chem. Commun.

Methods Enzymol.

Regular Article
A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry☆