Insulin Methyl Ester

There is considerable experimental evidence in support of the view that, in solution, the state of molecular dispersion of insulin is dependent on the net charge of the protein (10, 6, 8). In these investigations, alterations of the net positive charge were obtained by varying the pH of the solutions, with due consideration of the effect of the specific binding affinity of the protein for anions other than hydroxyl ions (8). In the present investigation, an attempt was made to vary the net positive charge of the protein by chemical modification of specific ionizable groups. Among the various possibilities which exist, esterification of the carboxyl groups with acid methanol appeared most promising, since insulin is particularly resistant to denaturation by acids or alcohol. The reaction of several proteins with acid methanol was studied by Fraenkel-Conrat and Olcott (7) who reported that, under specified conditions, complete esterification of the free carboxyl groups of proteins can be obtained without introducing other perceptible chemical modifications. This has been confirmed in the present work but it was found that in the case of insulin rigid control of the reaction with respect to time, temperature, and composition of the esterifying agent is of prime importance for the chemical, biological, and physical properties of the modified protein. The present paper describes the preparation of insulin methyl ester and the effect of methylation on the titration curve, solubility, and biological activity of the protein, whereas in subsequent papers of this series the effect of methylation on the molecular-kinetic properties of the protein will be described.