Precipitation of insulinotropin in the presence of protamine: effect of phenol and zinc on the isophane ratio and the insulinotropin concentration in the supernatant

Abstract

Purpose: The purpose of this study is to determine the minimum quantity of protamine required for the completion of insulinotropin precipitation under different precipitation conditions.

Methods: The experiments involved combining insulinotropin with varying concentrations of protamine in pH 7.2 phosphate buffered saline and analyzing the concentrations of both proteins in the supernatant. Isophane ratio (the protamine/insulinotropin molar ratio that results in a minimum total protein concentration in the supernatant) was determined for different precipitation conditions.

Results: When neutral solutions of insulinotropin (pI 5.3) and protamine (pI 13.8) were combined, precipitation did not occur. However, in the presence of phenol and/or zinc, insulinotropin co-precipitated with protamine. In the presence of phenol, the isophane ratio and the insulinotropin concentration in the supernatant were determined to be 0.08 and 0.18 mg/ml, respectively. In the presence of zinc, the isophane ratio increased with zinc concentration, apparently from the precipitation of protamine in the presence of zinc. The isophane ratio and the insulinotropin concentration in the supernatant were 0.13 and 0.13 mg/ml, respectively, when the zinc/insulinotropin molar ratio was one. In the presence of phenol and zinc with the zinc/insulinotropin molar ratio of 1.0, the isophane ratio and the insulinotropin concentration in the supernatant were 0.11 and 1 microgram/ml, respectively.

Conclusions: A method to determine the isophane ratio of protamine/insulinotropin precipitation was developed to determine the minimum quantity of protamine required for the completion of insulinotropin precipitation under different precipitation conditions. A synergistic effect between phenol and zinc on the precipitation of insulinotropin in the presence of protamine was found.