Vlasova T N, Leontieva O V, Ugarova N N
Faculty of Chemistry, Lomonosov Moscow State University, Moscow, Russia.
Biochemistry (Mosc). 2006 May;71(5):555-9. doi: 10.1134/s0006297906050142.
Dissociation constants (Ks) in the pH range 6.5-9.0 for complexes of luciferin, dimethyloxyluciferin (DMOL), and monomethylluciferin (MMOL) with recombinant wild-type and mutant (His433Tyr) luciferases from the Luciola mingrelica firefly were determined by fluorescent titration. The protonated effectors were bound by the wild-type and mutant luciferases better than the nonprotonated ones. The affinity of DMOL for the mutant luciferase was higher than for the wild-type luciferase at alkaline pH, whereas the affinity of MMOL was higher at all pH values studied. The fluorescence emission and excitation spectra of DMOL and MMOL in buffer solution (pH 7.8) were obtained in the absence and presence of luciferase. The fluorescence maxima of DMOL and MMOL complexes with luciferase were 20 and 100 nm, respectively, shifted to shorter wavelengths as compared to the values in buffer solution. This was explained by nonspecific and specific influence of the protein microenvironment on the fluorescence spectra of DMOL and its specific influence on the MMOL fluorescence spectra.
通过荧光滴定法测定了荧光素、二甲基氧化荧光素（DMOL）和单甲基荧光素（MMOL）与米氏萤光虫重组野生型和突变型（His433Tyr）荧光素酶在pH值6.5 - 9.0范围内的解离常数（Ks）。质子化的效应物与野生型和突变型荧光素酶的结合比未质子化的效应物更好。在碱性pH条件下，DMOL对突变型荧光素酶的亲和力高于野生型荧光素酶，而在所有研究的pH值下，MMOL的亲和力都更高。在有无荧光素酶的情况下，获得了缓冲溶液（pH 7.8）中DMOL和MMOL的荧光发射光谱和激发光谱。与缓冲溶液中的值相比，DMOL和MMOL与荧光素酶形成的复合物的荧光最大值分别有20和100 nm的蓝移。这是由蛋白质微环境对DMOL荧光光谱的非特异性和特异性影响以及对MMOL荧光光谱的特异性影响所解释的。
