Unusual chemical properties of the amino groups of insulin: implications for structure–function relationship

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Abstract
The chemical properties of the 3 amino groups of [porcine Zn] insulin were obtained at 10 and 37.degree. C using the competitive labeling technique with acetic anhydride as the labeling reagent. At 10.degree. C, pK values of 7.9, 7.2 and 7.8 were found for the glycyl Al, phenylalanyl B1 and lysyl B29 amino groups. When compared with standard amino compounds by means of a Bronsted plot, the 2 amino-termini were super-reactive and the lysyl .epsilon.-amino group buried. In the presence of CO2 at physiological pH values, all 3 amino groups became much less reactive indicating that they had reacted to form carbamino derivatives. Above pH 8 the reactivities of the glycyl amino terminus and .epsilon.-amino group increase sharply indicating that insulin in undergoing a conformational change which is most likely a change in its association state. At 37.degree. C the amino groups do not titrate normally but exhibit sharp increases in reactivity over the physiological pH range with the midpoints in the pH reactivity profiles between pH values of 7.0 and 7.3. This behavior is interpreted as a rapid disaggregation of insulin to form monomers as a result of the ionization of the amino groups. At physiological pH and temperature all 3 amino groups are deprotonated.