DECAY-ASSOCIATED FLUORESCENCE-SPECTRA AND THE HETEROGENEOUS EMISSION OF ALCOHOL-DEHYDROGENASE
DECAY-ASSOCIATED FLUORESCENCE-SPECTRA AND THE HETEROGENEOUS EMISSION OF ALCOHOL-DEHYDROGENASE
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DOI：
10.1021/bi00262a024
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
BRAND, L
中科院分区：
文献类型：
--
作者：
KNUTSON, JR;WALBRIDGE, DG;BRAND, L
描述了使用纳秒时间分辨的荧光衰减数据来获得与衰减相关的荧光光谱的过程。可以提取混合物中两种或多种成分的单独荧光光谱，而无需事先了解其光谱形状或重叠程度。该程序对于消除混浊样品荧光光谱中的散射光伪影也很有价值。该方法用于分离马肝乙醇脱氢酶中2个色氨酸残基的重叠发射光谱。酶、NAD+ 和吡唑之间形成三元复合物导致总色氨酸荧光减少。两种色氨酸残基的排放量均减少。隐藏的色氨酸（残基 314）经历动态猝灭，光谱分布没有变化。在相同条件下，色氨酸（残基 15）的荧光强度降低，衰减时间没有变化，但发射光谱发生红移。当游离酶被酸变性（琥珀酸盐缓冲液，pH 4.1）时，色氨酸荧光强度也会降低。变性的酶保留足够的结构，以为不同的色氨酸残基提供不同的微环境，如双指数衰变和光谱移动发射光谱（通过衰变关联揭示）所反映的那样。讨论了该技术对于生物大分子微观异质性研究的价值。
A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. The individual fluorescence spectra of 2 or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the 2 tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. The emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.