Wasylewski Z, poloczek H, Wasniowska A
Department of Biochemistry, Jagiellonian University, Krakow, Poland.
Eur J Biochem. 1988 Mar 15;172(3):719-24. doi: 10.1111/j.1432-1033.1988.tb13948.x.
A new procedure is described for using fluorescence-quenching data of tryptophan residues in proteins to resolve their fluorescence emission spectra. In this concept the Stern-Volmer quenching plot is determined at each particular emission wavelength and iterative non-linear least-squares fitting procedure allowed to resolve the steady-state emission spectra into components. The resolved components, attributed to each of tryptophan residue, can be characterized by different accessibility to the quencher. The ability to resolve fluorescence emission spectra can be improved by using different kinds of efficient quenchers, which can selectively quench the emission of exposed or both exposed and buried fluorophores. The method was used to decompose emission fluorescence spectra in two-tryptophan-containing proteins; horse liver dehydrogenase, sperm whale apomyoglobin and metalloprotease from Staphylococcus aureus. The resolved spectra of alcohol dehydrogenase and metalloprotease are in excellent agreement with those previously obtained by single-photon counting or phase methods. The method presented here is technically simple and does not require expensive instrumentation.
本文描述了一种新方法，用于利用蛋白质中色氨酸残基的荧光猝灭数据来解析其荧光发射光谱。在此概念中，在每个特定发射波长下测定斯特恩-沃尔默猝灭图，并通过迭代非线性最小二乘法拟合程序将稳态发射光谱解析为多个组分。归因于每个色氨酸残基的解析组分可通过对猝灭剂的不同可及性来表征。通过使用不同种类的高效猝灭剂可以提高解析荧光发射光谱的能力，这些猝灭剂可以选择性地猝灭暴露的荧光团或暴露和埋藏的荧光团的发射。该方法用于分解含两个色氨酸的蛋白质的发射荧光光谱，即马肝脱氢酶、抹香鲸脱辅基肌红蛋白和金黄色葡萄球菌金属蛋白酶。乙醇脱氢酶和金属蛋白酶的解析光谱与先前通过单光子计数或相位方法获得的光谱非常吻合。本文提出的方法技术上简单，且不需要昂贵的仪器。
