Yesook Kim

The Application of Crystal Soaking Technique to Study the Effect of Zinc and Cresol on Insulinotropin Crystals Grown from a Saline Solution

AbstractPurpose. The purpose of this study is to investigate the effect of zinc and cresol on the structure of insulinotropin crystals. Methods. Insulinotropin crystals grown from a saline solution were treated with zinc and/or m-cresol using a crystal soaking technique. The effects of these additives on the crystal structure were investigated with powder X-ray diffraction, photomicrography, and differential scanning calorimetry. The molecular interaction between insulinotropin and m-trifluorocresol in solution was also studied by 19F NMR. Results. The data suggest that the original crystals grown from a saline solution have relatively weak lattice forces. After the addition of m-cresol to the suspension of the insulinotropin crystals, the crystals were immediately rendered amorphous. The m-cresol molecules which diffused into the crystals through solvent channels may have disturbed the lattice interactions that maintain the integrity of the crystal. In contrast, the zinc added to the suspension stabilized the crystal lattice so that the subsequent addition of m-cresol did not alter the integrity of the crystals. A marked increase in melting point (206° versus 184°) and heat of fusion (24.6 J/g versus 1.4 J/g) of the crystals was observed after the treatment with zinc. The solubility of the zinc treated crystals in a pH 7.1 phosphate buffered saline was 1/20 of that of the original crystals. Conclusions. When the insulinotropin crystals were treated with the additives using a crystal soaking method, the crystals underwent structural changes. Zinc stabilized the crystal lattice, and reduced the solubility of the peptide.

Precipitation of Insulinotropin in the Presence of Protamine: Effect of Phenol and Zinc on the Isophane Ratio and the Insulinotropin Concentration in the Supernatant

AbstractPurpose. 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 µg/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.