4-（2-吡啶偶氮）间苯二酚在大肠杆菌天冬氨酸转氨甲酰酶锌释放研究中的应用。

The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase.

作者信息

Hunt J B, Neece S H, Ginsburg A

出版信息

Anal Biochem. 1985 Apr;146(1):150-7. doi: 10.1016/0003-2697(85)90409-9.

Abstract

The metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) has been used at pH 7.0 to monitor the mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase and Zn2+ uptake by regulatory dimers upon displacement of the mercurial reagent with 2-mercaptoethanol. The release of Zn2+ (as reflected by a yellow to orange color change in PAR solutions) is linked to dissociation of the enzyme since the six Zn2+ bonding domains stabilize catalytic and regulatory chain contacts; the rebinding of Zn2+ produces enzyme assembly and a corresponding decrease in the amount of PAR-Zn2+ complex. Using greater than 10-fold PAR to free Zn2+ at pH 7.0, delta epsilon = 6.6 +/- 0.2 X 10(4) M-1 cm-1 at 500 nm (20 degrees C) for (PAR)2Zn2+ complex formation (beta'2 approximately equal to 10(12) M-1). In kinetic studies at pH 7.0, PAR (10(-4) M) has been used to measure the instantaneous concentration of Zn2+ released from micromolar quantities of protein; second-order k = 2 X 10(7) M-1 s-1 for forming the 1:1 PAR:Zn2+ complex. These properties of PAR-Zn2+ interactions make PAR a generally useful reagent for studying Zn2+ release from proteins.

摘要

金属显色指示剂4-(2-吡啶偶氮)间苯二酚（PAR）已被用于在pH 7.0的条件下，监测汞促进大肠杆菌天冬氨酸转氨甲酰酶释放锌离子以及在用2-巯基乙醇取代汞试剂后调节二聚体对锌离子的摄取情况。锌离子的释放（通过PAR溶液中从黄色到橙色的颜色变化反映）与酶的解离有关，因为六个锌离子结合结构域稳定了催化链和调节链的接触；锌离子的重新结合导致酶组装，并且PAR-锌离子复合物的量相应减少。在pH 7.0时，使用比游离锌离子浓度高10倍以上的PAR，在500 nm（20℃）下，形成（PAR）₂Zn²⁺复合物时的Δε = 6.6±0.2×10⁴ M⁻¹ cm⁻¹（β'₂约等于10¹² M⁻¹）。在pH 7.0的动力学研究中，PAR（10⁻⁴ M）已被用于测量从微摩尔量蛋白质中释放的锌离子的瞬时浓度；形成1:1 PAR:Zn²⁺复合物的二级反应速率常数k = 2×10⁷ M⁻¹ s⁻¹。PAR与锌离子相互作用的这些特性使PAR成为研究蛋白质中锌离子释放的一种普遍有用的试剂。