A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry.
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, Ni(2+) concentrations contaminating proteins eluted from Ni(2+) 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.
Getz
E B
EB
Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA.
Xiao
M
M
Chakrabarty
T
T
Cooke
R
R
Selvin
P R
PR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D027682:Cation Transport Proteins; D004229:Dithiothreitol; D004578:Electron Spin Resonance Spectroscopy; D006868:Hydrolysis; D007202:Indicators and Reagents; D008956:Models, Chemical; D015879:Myosin Subfragments / Q000378:metabolism; D010084:Oxidation-Reduction; D010720:Phosphines; D011817:Rabbits; D013439:Sulfhydryl Reagents
D000251:Adenosine Triphosphatases; D027682:Cation Transport Proteins; D004229:Dithiothreitol; D007202:Indicators and Reagents; D015879:Myosin Subfragments; D010720:Phosphines; D013439:Sulfhydryl Reagents; C080938:tris(2-carboxyethyl)phosphine; C022761:potassium transporting ATPase