Analytical Biochemistry 273, 73–80 (1999) Article ID abio.1999.4203, available online at http://www.idealibrary.com on A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry1 Elise Burmeister Getz,* Ming Xiao,† Tania Chakrabarty,† Roger Cooke,* and Paul R. Selvin†,2 *Department of Biochemistry and Biophysics, and Cardiovascular Research Institute, University of California, San Francisco, California 94143; and †Department of Physics and Biophysics Center, University of Illinois, Urbana, Illinois 61801 Received January 28, 1999 Preserving the reactive sulfhydryls of a protein in a The newly introduced sulfhydryl reductant tris(2- reduced state is critical to the maintenance of function carboxyethyl)phosphine (TCEP) is a potentially at- of many proteins. The most commonly used disulfide tractive alternative to commonly used dithiothreitol reductants are thiols themselves (1). The mechanism of (DTT). We compare properties of DTT and TCEP im- disulfide reduction by thiols is an exchange of the thio- portant in protein biochemistry, using the motor en- late anion (XS2), as shown in Reactions [1] and [2]. zyme myosin as an example protein. The reductants equally preserve myosin’s enzymatic activity, which is XS 2 1 RSSR 3 RSSX 1 RS 2 [1] sensitive to sulfhydryl oxidation. When labeling with extrinsic probes, DTT inhibits maleimide attachment XS 2 1 RSSX 3 XSSX 1 RS 2 [2] to myosin and must be removed before labeling. In contrast, maleimide attachment to myosin was The two most commonly used thiol reductants are achieved in the presence of TCEP, although with less 2-mercaptoethanol and dithiothreitol (DTT)3 (Fig. 1). efficiency than no reductant. Surprisingly, iodoacet- In the case of DTT, Reaction [2] is intramolecular and amide attachment to myosin was nearly unaffected by so involves the formation of two products from one either reductant at low (0.1 mM) concentrations. In reactant, with the DTT being converted to a stable electron paramagnetic resonance (EPR) spectroscopy cyclic disulfide. As a result, reduction of disulfide by utilizing nitroxide spin labels, TCEP is highly advan- DTT has an equilibrium constant of 1.3 3 104 (2), tageous: spin labels are two to four times more stable compared to an equilibrium constant close to unity for in TCEP than DTT, thereby alleviating a long-standing 1 monothiol reductants such as 2-mercaptoethanol. problem in EPR. During protein purification, Ni2 con- 1 However, disulfide reduction by thiols can be incon- centrations contaminating proteins eluted from Ni2 venient when reacting protein sulfhydryls with extrin- affinity columns cause rapid oxidation of DTT without sic probes. The –SH groups of the reductant compete affecting TCEP. For long-term storage of proteins, directly with those of the protein for attachment of TCEP is significantly more stable than DTT without thiol-reactive labels such as maleimide and iodoacet- metal chelates such as EGTA in the buffer, whereas amide derivatives. Therefore, thiol-based reductants DTT is more stable if metal chelates are present. Thus are typically removed before the protein is labeled. In TCEP has advantages over DTT, although the choice addition, the sulfhydryls of DTT readily reduce the of reductant is application specific. © 1999 Academic Press nitroxide spin probes used in electron paramagnetic resonance (EPR) spectroscopy, thus eliminating the 1 This work was supported by NIH Grants HL32145 (R.C.) and 3 Abbreviations used: DTT, dithiothreitol; TCEP, tris(2-caroxy- AR44420 (P.R.S.), and by Bank of America–Giannini Foundation thyl)phosphine; DTNB, 5,59-dithiobis(2-nitrobenzoic acid); NTB, 2-ni- and UC Presidents Postdoctoral Fellowships (E.B.G.). tro-5-thiobenzoate; HMM, heavy meromyosin; TMRIA, tetramethylrho- 2 To whom correspondence should be addressed at Loomis Labo- damine-5-iodoacetamide; TMRM, tetramethylrhodamine-5-maleimide; ratory of Physics, 1110 W. Green St., University of Illinois, Urbana SL, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperdinyl) maleimide; TBP, tribu- IL 61801. Fax: (217) 244-7187. E-mail: [email protected]. tylphosphine; EPR, electron paramagnetic resonance. 0003-2697/99 $30.00 73 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 74 BURMEISTER GETZ ET AL. FIG. 1. Sulfhydryl reducing agents. free radical that allows detection of probe orientation assays were performed using heavy meromyosin and mobility. A third problem is that DTT oxidation is (HMM), a proteolytic fragment of the motor protein catalyzed by ubiquitous metal ions, such as Fe31 and myosin. HMM is a good test case because its enzymatic 1 Ni2 (1, 3, 4), and so DTT is not stable in the reduced activity is affected by oxidation or modification of its form for long times. two most reactive sulfhydrals, Cys-697 and Cys-707, It may be possible to circumvent these problems by and there is a simple assay to determine its enzymatic using trialkylphosphines as the reducing agent. In activity (reviewed by Crowder and Cooke (10)). Specif- 1991, Burns et al. (5) described a convenient and large- ically, modification of either of these sulfhydryls atten- scale synthesis of tris(2-carboxyethyl)phosphine uates HMM’s K1-ATPase hydrolysis rate (11). (TCEP) (Fig. 1), and TCEP has been commercially available since 1992. In aqueous solutions, TCEP sto- ichiometrically and irreversibly reduces disulfides ac- METHODS cording to Reaction [3] (6, 7). Materials. DTT was purchased from Sigma (St. Louis, MO), and TCEP was purchased from Molecular ~CH2CH2COOH!3P: 1 RSSR Probes (Eugene, OR). Single isomers of both tetra- methylrhodamine-5-iodoacetamide (TMRIA) and tet- 1 H O 3 ~CH CH COOH! PAO 1 2RSH [3] 2 2 2 3 ramethylrhodamine-5-maleimide (TMRM) were pur- chased from Molecular Probes (Eugene, OR). Stock TCEP has been shown to be significantly more stable solutions of these dyes were dissolved at mM levels in than DTT at pH values above 7.5, and a faster and anhydrous dimethyl sulfoxide (Aldrich, Milwaukee, stronger reductant than DTT at pH values below 8.0 WI). 5,59-dithio-bis(2-nitrobenzoic acid) (DTNB), and (8). Thus TCEP is a useful reductant over a much N-(1-oxyl-2,2,6,6-tetramethyl-4-piperdinyl) maleimide wider pH range (1.5–8.5 (8)) than is DTT, although the (SL) were purchased from Aldrich. buffer composition, including the presence of phos- Protein preparation. Myosin was prepared from phates, can deleteriously affect TCEP stability (4, 5, 8). rabbit back and leg muscles (12) and stored at 230°C In addition, TCEP has been advertised as being unre- in 0.3 M NaCl, 10 mM TES, 0.25 mM DTT, 50% glyc- active with thiol-reactive compounds, thereby elimi- erol. Heavy meromyosin (HMM) was prepared from nating the need to remove it before labeling (9). To quantify the advantages, if any, of TCEP over skeletal myosin by standard methods (13, 14). Protein DTT, we compared these two reductants in several concentrations were determined using extinction coef- ficients of 2.39 3 105 M21 cm21 (myosin) or 2.35 3 105 applications related to protein biochemistry: (1) stabil- 21 21 ity at neutral pH, including in the presence of trace M cm (HMM) at 280 nm. 1 Ni21 at concentrations expected to contaminate pro- ATPase assays. For K -ATPase assays, HMM (10.0 teins eluted from Ni21-affinity columns; (2) the ability mM) was stored at room temperature in 50 mM KCl, 2 to preserve enzymatic activity, tested over a range of mM MgCl2, 1 mM EGTA, 50 mM Mops, pH 7.0. Sam- reductant concentrations which we find stabilizes en- ples were prepared with TCEP or DTT or no reductant. zymatic activity and which is widely used in biochem- ATPase activities were measured by determining the istry, 0.1–5.0 mM; (3) interference with attachment of rate of release of inorganic phosphate at 25°C (15). 1 labels to protein thiols; (4) reduction of nitroxide spin K -ATPase was assayed in 0.6 M KCl, 1.0 mM EDTA, probes; and (5) the ability to cause unwanted protein 50 mM Mops, pH 7.0. The reaction was initiated by degradation at elevated temperatures used in gel elec- addition of 1.0 mM ATP. At 20, 60, 120, 180, 240, and trophoresis preparations. The second and third of these 300 s, aliquots were quenched with 3.1% perchloric TRIS(2-CARBOXYETHYL)PHOSPHINE VS DITHIOTHREITOL AS REDUCTANTS 75 acid. The rate of ATP hydrolysis was constant during at 412 nm using a molar extinction coefficient of 14,150 this time. M21 cm21. Since DTNB is not stable at high pH, au- Maleimide and iodoacetamide labeling. In the ab- toreducing to yield the colored NTB it was necessary to sence of nucleotide, only one of myosin’s reactive eliminate the contribution of DTNB autoreduction to sulfhydryls, Cys-707, is easily modified (11, 16, 17). 412 nm absorption. A blank (no TCEP or DTT) mea- Because HMM is a dimer, there are two Cys-7079s sured immediately prior to each of the TCEP or DTT per HMM molecule. HMM at 10 mM in 50 mM KCl, 2 readings was subtracted from the reading. It was rou- tinely confirmed that the extent of DTNB autoreduc- mM MgCl2, 50 mM Mops, pH 7.0 was reacted over- night on ice with TMRIA or TMRM. Labeling ratios tion did not change during the (short) course of a TCEP (dye:Cys-707) ranged from 1.0 to 3.9 for TMRM (20 to or DTT reading by comparing the blank’s absorption 78 mM TMRM), and from 1.0 to 2.0 for TMRIA (20 to measured immediately prior to and following a TCEP 40 mM TMRIA). In all cases, reductant concentration or DTT reading. is in excess of label concentration so as to best detect The percentages of reductant oxidation presented in inhibition of labeling by reductant.