Kurzban G P, Gitlin G, Bayer E A, Wilchek M, Horowitz P M
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
J Protein Chem. 1990 Dec;9(6):673-82. doi: 10.1007/BF01024762.
Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20-28% of the initial fluorescence with (KSV + V) less than or equal to 0.55 M-1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka = 5 M-1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weber et al., 1989).
生物素结合使链霉亲和素的色氨酸荧光发射降低39%，发射峰从333纳米蓝移至329纳米，半高带宽从53纳米降至46纳米。生物素诱导的发射差光谱类似于中等极性色氨酸的光谱。链霉亲和素荧光可分为两个寿命类别：2.6纳秒（34%）和1.3纳秒（66%）。结合生物素后，寿命分别为1.3纳秒（26%）和0.8纳秒（74%）。生物素结合使平均荧光寿命从1.54纳秒降至0.88纳秒。生物素不会淬灭吲哚的荧光。荧光变化与生物素结合导致构象变化一致，该变化使色氨酸靠近链霉亲和素中降低量子产率和寿命的部分。丙烯酰胺对荧光的淬灭揭示了两类荧光团。分析表明，一个受屏蔽的组分占初始荧光的20 - 28%，其（KSV + V）小于或等于0.55 M-1。更易接近的组分主要是静态淬灭。在不同丙烯酰胺浓度下对荧光寿命的测量证实了强烈的静态淬灭。由于静态淬灭可能是由于丙烯酰胺与链霉亲和素结合，构建了一种用于检测丙烯酰胺结合的染料置换测定法。丙烯酰胺确实与链霉亲和素结合（Ka = 5 M-1），并且可能结合在生物素结合位点内。在没有生物素的情况下，链霉亲和素的荧光没有特别容易被碘化物接近的部分。在有生物素存在时，碘化物既不淬灭荧光也不改变发射光谱，并且丙烯酰胺的接近程度显著降低。我们提出，始终排列在生物素位点的三个色氨酸足够靠近结合位点表面，以至于会被结合的丙烯酰胺淬灭。这些色氨酸对碘化物有屏蔽作用，很可能是由于空间位阻或离子阻碍阻止其扩散到结合位点。生物素结合所赋予的大部分屏蔽作用可归因于对这些残基以及生物素结合时移入结合位点的第四个色氨酸的直接屏蔽，如X射线研究所示（Weber等人，1989年）。
