Studies on the Constitution of Insulin*

The study of the course of reduction of insulin by thioglycolic acid at about pH 2 has made possible the preparation in dry state of insulin samples of a varying degree of reduction (1). Chemical and physical measurements conducted on these preparations demonstrated that incipient denaturation of the protein occurs when the reduction has proceeded for 150 minutes under the experimental conditions selected. The product corresponding to this reduction period has been designated as reduced native insulin. The sulfhydryl content of these preparations indicates that a reduction of one or two disulfide groups of the hormone has occurred. Furthermore, the pharmacodynamic activity has decreased approximately 50 per cent. These results suggested the working hypothesis that one or two dithio (-S-S-) linkages in insulin represent activating groups; i.e., regulate the functioning of the active center, the nature of which is still obscure. The experiments described in the present communication were designed to test the validity of this hypothesis. The reduced native insulin preparations have been studied in detail with respect to their chemical and physical properties. If the hypothesis is tenable, it would be expected that such experiments will not reveal any significant alterations in the properties of the hormone molecule other than the previously reported formation of a small number of free -SH groups. It is realized that the 50 per cent loss in physiological activity of the reduced insulin preparations