The chemical properties of the functional groups of insulin were determined at a concentration (0.5 microM) where the predominant species of insulin is the free (unassociated) monomeric unit. The glycine N-terminus and the four tyrosine phenolic groups had the same properties as in the associated forms of insulin. On the other hand the lysine epsilon-amino group and the two histidine imidazole groups had substantially altered properties. Some alteration in the properties of the phenylalanine N-terminus was also observed. The reactivity-pH profile for the imidazole groups showed a second ionization with a pKa of 10.1 in addition to an ionization with a pKa of 6.8. On the basis of the X-ray-crystallographic structure of hexameric insulin the observed changes can be accounted for by disruption of monomer-monomer or dimer-dimer interactions in the associated states of insulin. It is concluded that the conformation of the monomeric unit of insulin is essentially the same in its free and associated states in solution.

The method of competitive binding [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] with 1-fluoro-2,4-dinitrobenzene as the labelling reagent [Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175] was used to determine the chemical properties, namely pK and reactivity, of the amino groups, the histidine residues and the tyrosine residues of the dimeric form of pig zinc-free insulin at 20.0 degrees C. The N-terminal glycine residue of the A-chain has a pK of 7.7 and a slightly higher than normal reactivity. The N-terminal phenylalanine residue of the B-chain has a pK of 6.9 and is approximately an order of magnitude more reactive than a corresponding amino group with the same pK value. The lysine epsilon-amino group has an unusually low pK of 7.0 but has approximately the expected reactivity of such a group. In the case of the two histidine and four tyrosine residues only the average properties of each class were determined. The histidine residues have a pK value of approx. 6.6, but, however, their reactivity is at least an order of magnitude greater than that of a free imidazole group. The tyrosine residues have a pK value of approx. 10, but their average reactivities are substantially less than for a free phenolic group. At alkaline pH values above 8 the reactivity of all the functional groups show sharp discontinuities, indicating that insulin is undergoing a structural change that alters the properties of these groups.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.

No abstract

An N-terminal histidine residue of a protein or peptide has two functional groups, viz., an alpha-amino group and an imidazole group. A new procedure, based on the competitive labeling approach described by Duggleby and Kaplan [Duggleby, R. G., & Kaplan, H. (1975) Biochemistry 14, 5168-5175], has been developed by which the chemical reactivity of each functional group in such a residue can be determined as a function of pH. Only very small amounts of material are required, which makes it possible to determine the chemical properties in dilute solution or in proteins and polypeptides that can be obtained in only minute quantities. With this approach, the reactivity of the alpha-amino group of histidylglycine toward 1-fluoro-2,4-dinitrobenzene gave an apparent pKa value of 7.64 +/- 0.07 at 37 degrees C, in good agreement with a value of 7.69 +/- 0.02 obtained by acid-base titration. However, the reactivity of the imidazole function gave an apparent pKa value of 7.16 +/- 0.07 as compared to the pKa value of 5.85 +/- 0.01 obtained by acid-base titration. Similarly, in glucagon and vasoactive intestinal peptide (VIP), apparent pKa values of 7.60 +/- 0.04 and 7.88 +/- 0.18, respectively, were obtained for the alpha-amino of their N-terminal histidine, and pKa values of 7.43 +/- 0.09 and 7.59 +/- 0.18 were obtained for the imidazole function.(ABSTRACT TRUNCATED AT 250 WORDS)