辅酶NADH与色氨酸共振能量转移的荧光动力学研究

Fluorescence Resonance Energy Transfer Between Coenzyme NADH and Tryptophan

采用时间相关单光子计数技术,结合紫外-可见吸收光谱和稳态荧光光谱,对不同环境下的色氨酸和辅酶还原型烟酰胺腺嘌呤二核苷酸(NADH)之间的共振能量转移荧光动力学进行了研究.单体色氨酸、牛血清白蛋白以及乳酸脱氢酶蛋白与NADH之间相互作用的光谱数据表明,只有存在NADH结合位点的乳酸脱氢酶和NADH之间发生了荧光共振能量转移.进一步通过加入丙酮酸来阻断乳酸脱氢酶和NADH之间的荧光共振能量转移通道,时间分辨荧光光谱和衰减相关光谱(DAS)证实,蛋白结合位点的存在是NADH和色氨酸之间发生荧光共振能量转移的前提条件.DAS揭示了乳酸脱氢酶平均荧光寿命的减小主要是源于色氨酸中7.35 ns的荧光寿命成分与NADH之间的荧光共振能量转移,同时给出了NADH和色氨酸之间的能量转移效率,为研究NADH和蛋白之间的相互作用提供了新思路.

Fluorescence resonance energy transfer(FRET)between coenzyme nicotinamide adenine dinucleotide(NADH)and tryptophan in different environments were investigated by using time correlation single photon counting technique,combined with UV-Vis absorption and steady-state fluorescence spectroscopy.Single tryptophan,bovine serum albumin(BSA)and lactate dehydrogenase(LDH)were mixed with NADH,respectively,and the spectral data indicated that the energy transfer between tryptophan and NADH occurred only when lactate dehydrogenase was mixed with NADH.Pyruvic acid were added to block the FRET channel between lactate dehydrogenase and NADH,it was verified that the presence of protein-NADH binding sites was a prerequisite for FRET between NADH and tryptophan by time-resolved fluorescence spectroscopy and decayassociated spectra(DAS).Furthermore,the average fluorescence lifetime of tryptophan in lactate dehydrogenase was deceased and it was mainly due to the energy transfer between the tryptophan component with lifetime ofτ=7.35 ns and NADH,and the efficiency of energy transfer between NADH and tryptophan was calculated.The results might provide a new idea for further study of the interaction between NADH and proteins.