10] Reductive cleavage of cystine disulfides with tributylphosphine

Abstract

Publisher Summary To study the applicability of the tributylphosphine reduction to general protein chemistry, this chapter presents work on highly purified preparations of soluble proteins with known structure. All peptides and proteins studied [(Lys δ )-vasopressin, insulin, human serum albumin, and bovine ribonuclease] in the chapter can be fully reduced with a 5-20% molar excess of tributylphosphine with PBu 3 . No strong denaturing agent, such as urea or guanidinium chloride, seems to be necessary, except when (partially) reduced proteins precipitate. The reduction is carried out best at room temperature at slightly alkaline pH in Tris buffer (0.1 M) or in bicarbonate (0.5 M). Reduction at acid pH can be performed, but is much slower and the resulting thiols are unreactive in respect to alkylation under acidic conditions. All examples given in the chapter are reductions and in situ alkylations. If reductions are to be performed with a very small excess of PBu 3 (5-20%), it is advisable to exclude oxygen completely. This can be achieved as follows: The protein is dissolved in a sealed flask connected with a vacuum pump and a nitrogen cylinder. The flask is evacuated with stirring or shaking and then filled with nitrogen. This procedure is repeated two or three times and reagents are added under positive nitrogen pressure.