AN ULTRACENTRIFUGE STUDY OF REDUCED INSULIN

Abstract

The importance of the intact, oxidized state of the sulfur linkages of insulin to the physiological activity of the hormone was first clearly demonstrated by du Vigneaud and coworkers (1).In this investigation, the insulin was converted into the sulfhydryl form by treatment in neutral solution with an excess of cysteine or glutathione.It was found that the reduction invariably led to inactivation, but that subsequent oxidation was not followed by reactivation.Wintersteiner (2), in an extension of this work, found that the inactivation.of insulin was complete when about onethird of the total sulfur was reduced.The rate of change of the two processes indicated, however, that actually the reduction of a very small proportion of the disulfide groups in the insulin molecule was sufficient to cause extensive inactivation.Freudenberg and Wegmann (3) have reported more recently that reduced insulin may be partially reactivated by treatment with oxygen or hydrogen peroxide in the presence of an excess of cysteine or glutathione.This was explained on the hypothesis that the insulin molecule consists of a polypeptide chain along which small branches are bridged by way of disulfide linkages, and that reactivation of the reduced insulin in the presence of high concentrations of cysteine or glutathione was possible because of the substitution of these units for the original branch chains.Stern and White (4) found that under certain conditions of reduction an insulin preparation might be obtained which contained around 5 per cent of its total sulfur in the reduced form and which possessed 50 per cent of the biological potency of the untreated protein.In the present study we wish to obtain more definite evidence as to whether the insulin molecule actually is broken into smaller fragments when the disulfide linkages of the protein are cleaved by reduction.If the insulin molecule contains twenty-four disulfide linkages, as may be calculated from the cystine content (5) and the molecular weight (6), and these linkages are spaced at equal intervals in a polypeptide chain structure, one would expect complete reduction to produce fragments possessing molecular weights of around 2000.If the insulin molecule consists of a bundle of parallel polypeptide chains, any two adjacent ones of which are * Rockefeller Fellow, Upsala, Sweden.