Selective inactivation of NADPH-cytochrome P-450 (c) reductase by diazotized 3-aminopyridine adenine dinucleotide phosphate

Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.