Tris(3-hydroxypropyl)phosphine (THPP) and Tris(2-carboxyethyl)phosphine, hydrochloride (TCEP) are water soluble compounds that are used as neutral sulfhydryl reducing agents. They have a greater reducing capacity than DTT. In addition, THPP and TCEP are suitable for use in immobilized metal affinity chromatography because they do not reduce the metals involved.
To maintain biological activity in biological systems, it is important that the thiol groups in peptides and proteins remain in a reduced state. Thiols, such as 2-mercaptoethanol and DTT, are commonly used as disulfide reductants. However, DTT can interfere in the desired reactions of SH groups and it can also be oxidized by metal-affinity columns. Therefore it must be removed prior to peptide modification or purification.
On the other hand, trialkylphosphines exhibit high selectivity for disulfide bonds, but are generally not water soluble. However, the high water solubility of THPP and TCEP make these two phosphines ideal alternatives to DTT. Unlike DTT, they generally do not need to be removed prior to thiol modification. THPP has the added advantage of being effective over a wide pH range.1
THPP and TCEP are used to break disulfide bonds within and between proteins in a wide range of biological applications. These applications include protein cleavage and/or precipitation for molecular diagnostics, gene chips and microarrays. In fact, THPP and TCEP are both found in numerous enzyme kits for biological processing, such as protein extraction2, specific amino acid sequence cleavage3, and protein in-gel tryptic digestion.4
THPP is also useful for the removal of protecting groups from S-protected cysteine, the deoxygenation of sulfoxides, N-oxides, and other sulfur and nitrogen compounds5. It is also a reagent for peptide synthesis, particularly in Mitsunobu and Staudinger reactions6. TCEP, meanwhile, does not impede maleimide attachment, and is suitable as a reagent for the quantitative analysis of sulfides, disulfides, hypochlorite ions, iodine, iodate ions, sulfoxides, N-oxides, azides7, and the selective reduction of disulfides for organic synthesis.8
1. US Patent Application 60/469,821.
2. Novagen Protocol TB245 Rev. E 0304, BugBusterstrem_com Protein Extraction Reagent; Novagen User Protocol TB316 Rev. B 0804, YeastBusterTM Protein Extraction Reagent.
3. Takara HRV 3C Protease Product Manual v201304Da.
4. Agilent 5188-2749Protein In-Gel Tryptic Digestion Kit Instructions.
5. G.A. Olah, B.G.B. Gupta, S.. Narang, Synthesis 1978, 137.
6. (a). I. Bosch, F. Urpf, J. Vilarrasa, Chem. Commun., 91, 1995; (b) H. Fuwa e.a., Tetrahedron Lett., 2004, 45, 2323; J. S. Davies, J. Peptide Sci., 2003, 9, 471.
7. A.-M. Faucher, C. Grand-Maitre, Synth. Commun., 2003, 33, 3503.
8. J.A. Burns, J.C. Butler. J. Moran, G.M. Whitesides, J. Org. Chem., 1991, 56, 2648.
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Keywords: Deprotection, peptide, protein, phosphine, Mitsunobu, Staudinger, sulfhydryl reducing agents, metal affinity chromatography