The Photochemical Binding of Tetrachlorosalicylanilide to Bovine Insulin

3,3´4´5-Tetrachloro[ 14 C]salicylanilide (T 4 CS) was found to bind covalently to bovine insulin on irradiation with ultraviolet light (360 nm). The [ 14 C]T 4 CS-labelled insulin was reduced and carboxymethylated and then fractionated into A and B chains. The [ 14 C]T 4 CS was found to be exclusively linked to the insulin B chain. Digestion of the [ 14 C]T 4 CS-labelled B chain with trypsin followed by ionexchange chromatography, resulted in the isolation of a peptide containing 78% of the applied radioactivity. Amino acid analysis of this peptide showed it to consist of residues 1—22 of the insulin B chain with one histidine residue missing, linked to a single molecule of T 4 CS. Amino acid analysis of B chain from insulin labelled with 1.59 mol of T 4 CS after extensive irradiation, showed loss of phenylalanine in addition to the loss of one histidine residue, suggesting the secondary involvement of the B chain TV-terminal residue. The photobiochemical reaction of T 4 CS with bovine insulin takes place primarily with one of the histidine residues of the B chain.