The influence of carrier iodide, iodine monochloride and pH on the labelling of ox insulin with (131)I by the iodine monochloride method have been studied. 2. The quantitative effect of the iodide in the radioactive iodine preparation was that predicted from a calculation of its specific activity. No other interfering factors were detected in the [(131)I]iodide solutions used. 3. Increasing the molar ratio of iodine monochloride to insulin resulted in an increase followed progressively by a decrease in the proportion of (131)I bound, while the total iodine bound increased to an amount characteristic of pH and thereafter remained constant. 4. The influence of pH on the iodination of insulin with iodine monochloride was complex and the pH curve showed two maxima, at pH2.8 and 6.4. At pH2.8 it was not possible to exceed 8 atoms of iodine bound per molecule by increasing the molar ratio of iodine monochloride. Similarly, at pH6.4 the substitution value of 11.5 atoms of iodine per molecule could not be exceeded. 5. Iodinated insulins containing an average of 1.96, 2.74, 6.0 and 7.0 atoms of iodine per molecule fully retained the ability to bind guinea-pig anti-(ox insulin) serum, and the ability to compete with unlabelled insulin for antibody sites only became significantly changed in the most highly substituted preparations and in the presence of large concentrations of unlabelled insulin. 6. The method for the iodination of insulin with 98% incorporation of (131)I by using chloramine-t is described. 7. (131)I-iodinated insulin prepared with graded quantities of chloramine-t in excess of that required for efficient labelling was less efficiently bound by guinea-pig anti-(ox insulin) serum than insulin labelled by the iodine monochloride method.
