Analysis for disulfide bonds in peptides and proteins

This chapter discusses the analysis for disulfide bonds in peptides and proteins. Disulfide bonds make major contributions to the stability of the native conformations of proteins. The determination of the number of disulfide bonds per molecule is therefore crucial to structural studies of proteins. Many methods for the determination of disulfide bond concentration are proposed. Recently, a new method involving the use the reagent 2-nitro-5-thiosulfobenzoate (NTSB) is introduced. This method has significant advantages, the two most important being that it is both sensitive and quantitative. The reaction of NTSB with thiols and disulfides can be carried out in the presence of both dissolved oxygen and the reducing agent, sodium sulfite, which eliminates the inaccuracy and inconvenience associated with the necessity to work under an oxygen-free atmosphere as well as the need to remove the reducing agent. The NTSB assay is actually composed of two sequential reactions. The first reaction is the cleavage of a disulfide bond with sodium sulfite. The second reaction involves the nucleophilic attack of the thiolate produced in first reaction on NTSB to yield 1 mol each of a thiosulfonate and 2-nitro-5-thiobenzoate (NTB).