Labeling of insulin with non-radioactive 127I and application to incorporation of radioactive 125I for use in receptor-binding experiments by high-performance liquid chromatography.

Conditions for the labeling of insulin with radioactive iodine isotopes were investigated by means of incorporation of non-radioactive 127I into the peptide. Either the chloramine-T (CT) or lactoperoxidase-hydrogen peroxide (LPO) technique was applied and reversed-phase high-performance liquid chromatography (RP-HPLC) was used for analysis of the reaction products. The LPO method provided the 127I-labeled peptide within 15-30 min, whereas the CT alternative yielded the labeled substrate even within 15 s. However, the latter reaction can only be controlled in a reproducible manner with difficulty and undesired side-reactions became increasingly prominent when the reaction time of 15 s was exceeded for only a few seconds. In another experiment, the LPO technique was applied for radiolabeling insulin with 125I. The product was first purified by size-exclusion chromatography (SEC) and then subjected to RP-HPLC. SEC yielded two peaks. The smaller one, which eluted at a slightly higher Kd value (accounting for about 14% of total radioactivity) predominantly consisted of material eluting at the column's void volume under the conditions of RP-HPLC, whereas the main SEC fraction (accounting for about 86% of total radioactivity) yielded a single peak, as shown by HPLC. The radioactive material attributable to the main SEC fraction revealed the expected receptor-binding properties, as evidenced by displacement experiments with non-radioactive insulin, as well as the action of tetradecanoyl phorbol acetate on the binding characteristics and thus indicating formation of a labeled hormone retaining biological activity.