Chemical properties of the functional groups of insulin

Abstract

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.

Solid-state Stability of Human Insulin II. Effect of Water on Reactive Intermediate Partitioning in Lyophiles from pH 2–5 Solutions: Stabilization Against Covalent Dimer Formation

“…The preference for covalent dimerization in the amorphous solid state at high water content (i.e., rubbery state) may reflect the higher insulin:water concentration ratio in amorphous solids at high water content in relation to that in concentrated solutions. This difference, coupled with the substantially greater nucleophilicity of amines compared with water and the relatively low pK a values of the amino groups involved (Phe B1 pK a ) 7.1 or less; Gly A1 pK a ) 8.0 [49][50][51] ) should lead to a preference for covalent dimerization as 'pH' approaches the pK a values of the amines. The higher prevalence of [Asp A21 -Gly A1 ] dimers in the solid-state degradation, despite the full unit higher pK a of Gly A1 in comparison with Phe B1 and the predominance of the [Asp A21 -Phe B1 ] dimers in solution, was unexpected.…”

Structure-function relationships in the free insulin monomer

“…the reactivity relative to that of a standard amine with the same pKa value that lies on the plot (Kaplan et al, 1971). Table 1 summarizes the pKa values and relative reactivities obtained for the amino groups in insulin in its free monomeric (present study) and associated forms (Sheffer & Kaplan, 1979;Chan et al, 1981). It is clear that these parameters are similar for the glycine N-terminus in all three studies but different for the lysine and phenylalanine amino groups.…”

“…Comparison of this parameter for the histidine imidazole function with those of standard imidazole groups (Cruickshank & Kaplan, 1975) shows that at least one of the two histidine residues in the insulin free monomer has higher than 'normal' reactivity. Comparison of the average rate constant for the four tyrosine residues (k = 2.7 M-1 min-') with that of the phenolic hydroxyl group of N-acetyltyrosine amide (k = 29 M-1 min-'; Chan et al, 1981) shows that these residues have a much lower-than-expected reactivity.…”

“…, both curves together. lie on a Br6nsted plot for the reaction of a series of standard amines with Dnp-F (Chan et al, 1981), a second-order pH-dependent rate constant of 0.615 M-1 * min-' is calculated. By using this value the second-order rate constants for the reactions of the functional groups in monomeric insulin with Dnp-F were estimated.…”

“…The solutions from step (5) (Scheme 1) were extracted with diethyl ether (4 x 5 ml) to remove [3H,'4C]Dnpalanylalanine and dinitrophenol as well as to aid in the precipitation of the protein. The [3H, 4C]Dnp derivatives were isolated from the precipitated protein solution after hydrolysis in 6 M-HCI and purified as described previously (Chan et al, 1981). The [3H,14C]Dnp-alanylalanine was separated from dinitrophenyl by the method of Steven (1962) and purified by t.l.c.…”