| Abstract: | As a means for probing the microenvironment of zinc in the insulin hexamer and to investigate the effects of calcium ion on the assembly and the structure of the two-zinc insulin hexamer, the thermodynamics and kinetics of the reaction between the chromophoric divalent metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) and zinc-insulin have been investigated over a wide range of conditions. For PAR]0 much greater than Zn2+]0 and Zn2+]/In] less than or equal to 0.33, the reaction leads to the sequestering and ultimate removal of all of the insulin-bound Zn2+; for Zn2+]0 much greater than PAR]0, two stable ternary complexes are formed where Zn2+ has ligands derived from PAR as well as from hexameric insulin. For Zn2+]/In] ratios below 0.33, the equilibrium distribution between the two ternary complexes is dependent on the Zn2+]/In] ratio. One of the complexes is assigned to the monoanion of PAR coordinated to Zn2+ that resides in a His-B10 site. The other complex is proposed to involve the coordination of (PAR)Zn to the site formed by the alpha-NH2 group of Phe-B1 and the gamma-carboxylate ion of Glu-A17 across the dimer-dimer interface on the surface of the hexamer. With either PAR or zinc-insulin in large excess, the kinetics of the PAR optical density changes are remarkably similar and biphasic. The faster step is first order in PAR and first order in insulin-bound Zn2+ (k congruent to 3 X 10(3) M-1 s-1) and involves the formation of an intermediate in which PAR is coordinated to insulin-bound zinc at the His-B10 site.(ABSTRACT TRUNCATED AT 250 WORDS) |
