1955 Bischoff Reaction Products of Insulin in Urea Solution

On the basis of differences in solubility and bioassay by the hypoglycemic response, five reaction products were isolated when amorphous or crystalline insulin was permitted to react in concentrated aqueous urea solution under various conditions of temperature and time. Insulin OU is formed when the insulin is precipitated immediately, from the urea solution, by the addition of a large volume of water. It is insoluble at pH 7.0 and its suspension has a delayed biologic response while its acid solution has a normal response. Insulin IU is formed when the reaction in urea solution is prolonged. It is soluble at pH 6.5 and has the same biologic response as regular insulin. Insulin IIU, the next reaction product, is insoluble in 0.03 N sulfate at PH 3, is soluble at the PH of the body fluids and has a delayed biologic response even when given intravenously. Its formation follows fist-order kinetics. The energy of activation is about 33,000 cal. per mole, the free energy of activation 26,000 cal. per mole at 38′ and the entropy change of activation 18-27 cal. per degree per mole. Insulin IIIU, formed on further prolonging the time in urea solution, differs from insulin IIU in that it is insoluble at pH 2.0, dilute HCI, and has half the biologic activity of insulin IIU. Insulin IVU, the next reaction product, is devoid of biologic activity. When heated at 99O, pH 2.0, insulin IIU forms both the classic heat precipitate and a precipitate soluble at pH 8.4. The heat precipitate of insulin dissolves in concentrated urea solution at 99O, thereby restoring its biologic activity.


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