A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry1

t t d The newly introduced sulfhydryl reductant tris(2arboxyethyl)phosphine (TCEP) is a potentially atractive alternative to commonly used dithiothreitol DTT). We compare properties of DTT and TCEP imortant in protein biochemistry, using the motor enyme myosin as an example protein. The reductants qually preserve myosin’s enzymatic activity, which is ensitive to sulfhydryl oxidation. When labeling with xtrinsic probes, DTT inhibits maleimide attachment o myosin and must be removed before labeling. In ontrast, maleimide attachment to myosin was chieved in the presence of TCEP, although with less fficiency than no reductant. Surprisingly, iodoacetmide attachment to myosin was nearly unaffected by ither reductant at low (0.1 mM) concentrations. In lectron paramagnetic resonance (EPR) spectroscopy tilizing nitroxide spin labels, TCEP is highly advanageous: spin labels are two to four times more stable n TCEP than DTT, thereby alleviating a long-standing roblem in EPR. During protein purification, Ni conentrations contaminating proteins eluted from Ni ffinity columns cause rapid oxidation of DTT without ffecting TCEP. For long-term storage of proteins, CEP is significantly more stable than DTT without etal chelates such as EGTA in the buffer, whereas TT is more stable if metal chelates are present. Thus CEP has advantages over DTT, although the choice f reductant is application specific. © 1999 Academic Press