Practical Protein Chemistry - A. Darbre 1989

Disulfide Bonds
Special Methods
Specific Cleavage at Cysteine Residues

Highly Structure/129.html">Specific Methods include Cysteine residue Cleavage using 2-nitro-5-thiocyanobenzoic acid (NTCB) [25]. The efficacy of this method was demonstrated using Histocompatibility Antigens, where adjacently positioned disulfide loops were compared using this technique [15]. The sizes of the disulfide loops were determined from the lengths of the resulting fragments. This method can be applied to identify structural Homology among related Proteins. Unfortunately, the resulting fragments feature N-terminal thiazolidine rings and cannot be sequenced directly. Evidence suggests that deblocking can be performed using Raney nickel [32] (Chapter 2). In this process, cystine residues are converted to Alanine and Methionine residues to β-aminobutyric acid, thereby making sequencing possible.

4.7.3.1. Cleavage with 2-nitro-5-thiocyanobenzoic acid [10, 15]. A sample of lyophilized protein (a few nmol) is dissolved in 0.2 M Tris-acetate buffer (pH 8.0) containing 6 M guanidine-HCl + 2 mM dithiothreitol + 1 mM EDTA, and incubated at 37 °C for 2 h. The sample is then diluted with 0.7 ml of the original buffer, and 0.5 ml of 30 mM NTCB is added. The reaction mixture is incubated at 20 °C for 30 min, adjusted to pH 9.5 with 1 M NaOH, and incubated again at 55 °C for 24 h. The reaction is quenched by acidification to pH 4 with glacial acetic acid.