13] Cleavage at cysteine after cyanylation

Abstract

Publisher Summary The SH groups of denatured proteins can be converted quantitatively to SCN groups with 2-nitro-5-thioeyanobenzoate (NTCB) 1,2, or with other reagents. NTCB is readily synthesized and can be labeled with [ 14 C] cyanide. Exposure to pH 8-9 results in cleavage at the amino group of the modified cysteine residue in excellent yield. Because disulfides do not react with NTCB, selective cleavage at cysteine can be obtained in the presence of cystine on the basis of this reaction. Specific SH groups of undenatured papain, aspartate transcarbamylase, isocitrate dehydrogenase, aspartate aminotransferase, NAD-specific glutamate dehydrogenase, glyceraldehyde-3-P dehydrogenase, and myosin can be modified with NTCB, or with 5,5'-dithiobis plus cyanide. Specific cleavage at cysteine residues specifically modified in native proteins provides a convenient method for locating residues. The use of disulfides formed from the 6-thio analog of ATP followed by cyanide, or direct cyanylation of protein SH groups with a 6-thiocyano analog of ATP has led to the incorporation of label and specific clearage in several cases.