Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phase high-performance liquid chromatography.

The specificity and rate of cleavage of adrenocorticotrophic hormone (ACTH) peptide bonds by endoproteinase Arg C were analyzed using capillary electrophoresis (CE) and reversed-phase (C18) high-performance liquid chromatography (HPLC). Acidic cleavage products were readily resolved by CE in uncoated capillaries using low ionic strength electrolytes. However, products predicted to have a net positive charge greater than 2 or more than 4 positively charged groups per peptide did not migrate out from the capillary at low ionic strength. Addition of salts and zwitterions to the electrolyte decreased capillary-peptide interactions such that all of the ACTH peptides examined were eluted with high efficiency separation by CE. Commercially obtained endoproteinase Arg C preparations exhibited peptidase activity at Lys-15-Lys16 and at Lys16-Arg17 in addition to the expected cleavage at Arg-X bonds. ACTH peptide bond cleavage rates for Arg8-Trp9, Arg17-Arg-18, Lys15-Lys16, and Lys16-Arg17 were 1.46, 0.096, 0.57, and 0.029 mumol min-1 mg-1 respectively. CE separations generally exhibited better resolution and were accomplished in shorter times than C18 HPLC separations. These properties make CE a particularly appropriate method for kinetic analysis of proteolytic enzyme action on peptide substrates.

Introduction of sulfhydryl groups into proteins at carboxyl sites.

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.

N-iodoacetyltyramine: preparation and use in 125I labeling by alkylation of sulfhydryl groups.

Preparation and use of N-iodoacetyltyramine in generation of 125I-labeled compounds is described. The kinetics of alkylation of N-acetylcysteine by N-iodoacetyltyramine (k2 = 3.0 M-1 s-1) and N-chloroacetyltyramine (k2 = 0.12 M-1 s-1) indicate that N-iodoacetyltyramine is more useful for labeling sulfhydryl-containing compounds to high specific activity with 125I. Conditions for preparation of carrier-free 125I-labeled N-iodoacetyl-3-monoiodotyramine in 50% yield based on starting iodide are described. The high degree of group specificity of N-iodoacetyl-3-monoiodotyramine reaction with sulfhydryl groups is demonstrated by the high reactivity toward sulfhydryl-containing bovine serum albumin and low reactivity toward N-ethylmaleimide-blocked bovine serum albumin and IgG. 125I-labeled N-iodoacetyl-3-monoiodotyramine was also used to prepare an 125I-labeled ACTH derivative that retains full biological activity, further demonstrating the selectivity toward reactions with sulfhydryl groups.