REDUCTION AND REOXIDATION OF BOVINE PROINSULIN. EFFECT OF pH, ZINC IONS, TEMPERATURE AND CONCENTRATION

The influence of pH, temperature and the concentrations of zinc ions and reduced proinsulin upon the yields of reoxidized proinsulin were studied using the insulin radioimmunoassay. The optimal conditions were found to be pH 8.5, absence of zinc ions and a low protein concentration. However, even at a concentration of reduced proinsulin of 1.47 μM, the reoxidized proinsulin did not compete with 125I‐insulin for the insulin antibodies as strongly as did genuine proinsulin. At high concentrations of reduced proinsulin (14.7 μM), the yield increased with decreasing temperature; at a 10‐times lower concentration the temperature had no influence upon the yields. Adsorption of reduced prosinulin to glassware was either irreversible or had a denaturing effect rendering the products immunologically inactive. Spontaneous reoxidation of reduced proinsulin is a rather delicate process, and the optimal conditions are different from those present in vivo. When the reduction of proinsulin was carried out at a concentration of 31.5 μM, zinc ions were found to stabilize the reduced protein against denaturation and precipitation for more than 24 hours. However, at this concentration, reduced proinsulin is in a thermodynamic ally unstable state, both with and without zinc.