Kaarsholm N C, Dunn M F
Biochemistry. 1987 Feb 10;26(3):883-90. doi: 10.1021/bi00377a032.
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)
作为探测胰岛素六聚体中锌微环境以及研究钙离子对双锌胰岛素六聚体组装和结构影响的一种手段，在广泛的条件下研究了发色二价金属离子螯合剂4-(2-吡啶偶氮)间苯二酚（PAR）与锌-胰岛素之间反应的热力学和动力学。当[PAR]₀远大于[Zn²⁺]₀且[Zn²⁺]/[胰岛素]小于或等于0.33时，该反应导致所有与胰岛素结合的Zn²⁺被螯合并最终去除；当[Zn²⁺]₀远大于[PAR]₀时，形成两种稳定的三元复合物，其中Zn²⁺具有源自PAR以及六聚体胰岛素的配体。对于[Zn²⁺]/[胰岛素]比率低于0.33的情况，两种三元复合物之间的平衡分布取决于[Zn²⁺]/[胰岛素]比率。其中一种复合物被认为是PAR的单阴离子与位于His-B10位点的Zn²⁺配位。另一种复合物被认为涉及(PAR)Zn与由Phe-B1的α-NH₂基团和Glu-A17的γ-羧酸根离子在六聚体表面的二聚体-二聚体界面形成的位点配位。当PAR或锌-胰岛素大量过量时，PAR光密度变化动力学非常相似且呈双相。较快的步骤对PAR为一级，对与胰岛素结合的Zn²⁺为一级（k约为3×10³ M⁻¹ s⁻¹），涉及形成一种中间体，其中PAR在His-B10位点与与胰岛素结合的锌配位。（摘要截断于250字）
