A Tb~(3+)-Tiron Complex Fluorescence Quenching Method for Determination of NADP
- 化学化工－已发表论文 
辅酶与酶蛋白组成结合蛋白酶,而结合蛋白酶催化反应的专一性取决于所含的辅酶,因此有必要建立辅酶的灵敏测定方法,并用于结合蛋白酶的催化动力学行为、构象及活性等化学特性的表征.实验表明辅酶II显著猝灭铽钛铁试剂络合物的荧光.本文推导了ΔF与辅酶II初始浓度的线性关系式(ΔF=nk[Q]0 ),并据此建立了辅酶II的高灵敏荧光分析方法.研究了荧光光谱、缓冲液、pH范围、铽离子和钛铁试剂最佳浓度、灵敏度、检测限和干扰等分析化学特性.辅酶II在40. 0nmol/L~1. 0μmol/L浓度范围内呈良好的线性,线性回归方程:ΔF=0. 868C(nmol/L) -13. 1(R2 =0. 994),检测限:1. 63×10-8 mol/L,且牛血清白蛋白及其它生物学中的常见离子在一定范围内均不干扰测定.结果表明,该方法用于测定辅酶II操作简便,测定快速,并预示着该方法可应用于若干含磷酸基的重要辅酶的测定.Conjugated proteases are composed of apoenzymes and coenzymes.The specificity of the enzyme catalysis is dependent on the coenzyme in the conjugated protease.It is necessary to develop a sensitive method for the determination of coenzymes,by which some chemical properties of conjugated proteases,such as its dynamics behavior,construction and activity,can be realized deeply.The experiment shows that NADP could remarkably quench the fluorescence intensity of Tb~(3+)-Tiron complex in Tris-HCl buffer solution of pH=7.60±0.01,and a sensitive fluorescence quenching method was developed for the determination of NADP.The quantitative formula was theoretical induced.It was demonstrated that the ΔF was directly proportional to and the beginning concentration of NADP(ΔF=nk[Q]_0).The analytical characteristics, such as fluorescence spectrum,buffer solution,pH range,optimum concentrations of Tb~(3+) and Tiron,sensitivity,detection limit and reference,etc.,were studied.A linear concentration range for NADP determination was obtained from 40.0 nmol/L to 1.0μmol/L,and its linear regression equation is ΔF=0.868C(nmol/L)-13.1 (R~2=(0.994)).The detection limit is high up to 1.63×10~(-8) mol/L.The bovine serum albumen and frequent ions in biology can’t interfere the determination of NADP within limits.The obtained results implied that the proposal method should find its application to the (determination) of other important coenzymes,which contain phosphates.