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 (TC EP) is a potentially attractive alternative to commonly used dithiothreitol (DTT). We compare properties of DTT and TCEP important in protein biochemi stry, using the motor enzyme myosin as an example protein. The reductants e qually preserve myosin's enzymatic activity, which is sensitive to sulfhydr yl oxidation. When labeling with extrinsic probes, DTT inhibits maleimide a ttachment to myosin and must be removed before labeling. In contrast, malei mide attachment to myosin was achieved in the presence of TCEP, although wi th less efficiency than no reductant. Surprisingly, iodoacetamide attachmen t to myosin was nearly unaffected by either reductant at low (0.1 mM) conce ntrations. 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-standin g problem in EPR. During protein purification, Ni2+ concentrations contamin ating proteins eluted from Ni2+ affinity columns cause rapid oxidation of D TT without affecting TCEP. For long-term storage of proteins, TCEP is signi ficantly more stable than DTT without metal chelates such as EGTA in the bu ffer, whereas DTT is more stable if metal chelates are present. Thus TCEP h as advantages over DTT, although the choice of reductant is application spe cific. (C) 1999 Academic Press.