Elongation of the interchain disulfide bridges of insulin. A synthetic analog.

The sythesis and isolation in purified form of an analog of insulin with the interchain disulfide bridges elongated by a methylene group is described. This analog differs from the parent molecule in that the cystein residues occupying positions A-7 and A-20 and involved in the formation of the two interchain disulfide bridges of insulin have been replaced by homocysteine residues. For the synthesis of this compound the Hcy-7, 20-A chain of sheep insulin was chemically synthesized and isolated in the S-sulfonated form. Conversion of the latter product to the sulfhydryl derivative and combination with the S-sulfonated form of the B chain of sheep insulin yielded the [Hcy-7, 20-A] insulin. Isolation of the analog from the combination mixture was effected by chromatography on a carboxymethylcellulose column with acetate buffer (pH 3.3) and an exponential sodium chloride gradient. This analog, by the mouse convulsion assay methods and in doses at least 40-fold higher than those normally used for insulin assay, was inactive. By the radioimmunoassay method this synthetic analog was found to possess a potency of 2 i.u./mg. It is concluded that the biological activity of insulin depends critically on a particular geometry conferred on the molecule by the proper placement of the A and B chains.