Monoiodoinsulin

The iodination of zinc-free insulin was catalyzed by lactoperoxidase, and monoiodoinsulin was purified from the reaction mixture by chromatography on DEAE-Sephadex (A-25). This fraction was demonstrated to be essentially pure by the criteria of polyacrylamide gel electrophoresis, specific radioactivity, and neutron activation analysis for iodine content. This derivative demonstrated the ultraviolet absorption characteristics expected of a monoiodotyrosyl insulin. Circular dichroic and immunological studies indicated no significant perturbations in the insulin molecule due to the attachment of iodine. Basic hydrolysis and subsequent amino acid analysis demonstrated that 85 to 90% of the total iodine was associated with 3-monoiodotyrosine, with the remaining iodine apparently associated with hydrolytic degradation products of 3-monoiodotyrosine. Oxidative sulfitolysis followed by enzymatic hydrolysis of the S-sulfo A and B chains indicated that this monoiodoinsulin was substituted predominantly (76%) on the tyrosine at position 14 of the A chain. The biological activity of monoiodoinsulin was indistinguishable from that of native insulin by the criterion of blood sugar depression in alloxan diabetic mice, and was 70 to 80% relative to insulin in the mouse convulsion assay. It therefore seems likely that the absolute integrity of tyrosine A14 is not required for the expression of the biological activity of insulin. A marked effect of zinc on both the immunological reactivity and near ultraviolet circular dichroism of insulin was noted during the course of this work. In addition, the effect of pH on the position of iodine substitution was examined, and a method is presented for the characterization of iodination reaction mixtures by polyacrylamide gel electrophoresis.