摘要
报道了Tb3+ - TR络合物探针用于研究脱氢酶,并建立了3种典型脱氢酶的定量测定方法,测定乙醇脱氢酶(ADH)、苹果酸脱氢酶(MDH)和葡糖- 6 -磷酸脱氢酶(G 6 PDH)的线性范围分别为: 2. 14×10-6 ~5. 50×10-5 g/mL、4. 17×10-6 ~1. 03×10-4 g/mL和5. 02×10-6 ~3. 51×10-5 g/mL;检测限分别为: 1. 02×10-7 g/mL、5. 32×10-7 g/mL和1. 25×10-6 g/mL.研究结果表明,Tb3+ TR络合物除可以作为脱氢酶定量测定的灵敏探针外,还可以用作指示乙醇脱氢酶(ADH)热变性过程的灵敏探针,探测结果显示ADH的热变性过程在50~60℃有一突变,从另一角度阐明了ADH的热变性过程不是长期以来一直认为的“全或无”的过程,而是一个构象渐变的过程.由于ADH分子是由酶蛋白和NAD+组成的全酶,其变性失活过程,除了与酶蛋白有关以外,还与NAD+有关.利用Tb3+ - TR络合物荧光探针可以较灵敏地监测到这一点.
The study on dehydrogenase with Tb^(3+)-TR complex as a fluorescence probe was reported.The determination methods of alcohol dehydrogenase(ADH),malate dehydrogenase(MDH) and glucose-6-phosphate dehydrogenase(G-6-PDH) were constructed.The linear ranges of ADH,MDH and G-6-PDH are 2.14×10^(-6)~5.50×10^(-5) g/mL,4.17×10^(-6)~1.03×10^(-4) g/mL and 5.02×10^(-6)~3.51×10^(-5)g/mL,respectively.The respective detection limits are 1.02×10^(-7) g/ml,5.32×10^(-7) g/mL and 1.25×10^(-6) g/mL,respectively.The results indicated that Tb^(3+)-TR complex can be utilized as a fluorescence probe not only to determine dehydrogenase,but also to monitor the denaturation and inactivation procedure of thermally denatured ADH.The monitoring results indicated that there was a special variety from 50~60℃ during the thermal denaturation process of ADH.The experiment offered another evidence to interpret the unfolding process of ADH molecule at different temperatures.It meant that the unfolding process of thermal denatured ADH was not an all-or-none process but a process of gradual changes,because ADH is composed of apoenzyme and NAD^+,the process of its denaturation and inactivation was associated not only with the apoenzyme but also with NAD^+,which can be monitored sensitively with the Tb^(3+)-Tiron complex as a fluorescence probe based on its fluorescence quenching by the phosphate moieties of ADH.
出处
《厦门大学学报（自然科学版）》
CAS
CSCD
北大核心
2005年第3期381-385,共5页
Journal of Xiamen University：Natural Science
