ABSTRACT
The chemical properties of the three amino groups of insulin were obtained at 10 and 37 degrees C using the competitive labelling technique with acetic anhydride as the labelling reagent. At 10 degrees C, pK values of 7.9, 7.2, and 7.8 were found for the glycyl A1, phenylalanyl B1, and lysyl B29 amino groups. When compared with standard amino compounds by means of a Brønsted plot, the two amino-termini were found to be 'super-reactive' and the lysyl epsilon-amino group buried. In the presence of carbon dioxide at physiological pH values, all three 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 is undergoing a conformational change which is most likely a change in its association state. At 37 degrees 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 behaviour is interpreted as a rapid disaggregation of insulin to form monomers as a result of the ionization of the amino groups. It is concluded that at physiological pH and temperature all three amino groups are deprotonated.
