Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.
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.
D000089:Acetic Anhydrides; D000596:Amino Acids / Q000032:analysis; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D004139:Dinitrofluorobenzene; D005898:Glass; D007328:Insulin / Q000302:isolation & purification; D008968:Molecular Conformation; D011137:Polystyrenes; D011138:Polytetrafluoroethylene / Q000031:analogs & derivatives
D000089:Acetic Anhydrides; D000596:Amino Acids; D007328:Insulin; D011137:Polystyrenes; C040378:poly(ethylene-tetrafluoroethylene); C031800:acetic anhydride; D011138:Polytetrafluoroethylene; D004139:Dinitrofluorobenzene