Abou-Zied Osama K, Al-Shihi Othman I K
Department of Chemistry, Faculty of Science, Sultan Qaboos University, P.O. Box 36, Postal Code 123, Muscat, Sultanate of Oman.
J Am Chem Soc. 2008 Aug 13;130(32):10793-801. doi: 10.1021/ja8031289. Epub 2008 Jul 19.
Subdomain IIA binding site of human serum albumin (HSA) was characterized by examining the change in HSA fluorescence in the native, unfolded, and refolded states. The study was carried out in the absence and presence of small molecular probes using steady-state and time-resolved fluorescence measurements. 2-Pyridone, 3-pyridone, and 4-pyridone bear similar molecular structures to those found in many drugs and are used here as probes. They are found to specifically bind in subdomain IIA and cause a reduction in the fluorescence intensity and lifetime of the Trp-214 residue in native HSA which is located in the same subdomain. The efficiency of energy transfer from Trp-214 fluorescence to the probes was found to depend on the degree of the spectral overlap between the donor's fluorescence and the acceptor's absorption. After probe binding in subdomain IIA, the distance between the donor and acceptor was calculated using Forster theory. The calculated quenching rate constants and binding constants were also shown to depend on the degree of spectral overlap. The results point to a static quenching mechanism operating in the complexes. Denaturation of HSA in the presence of guanidine hydrochloride (GdnHCl) starts at [GdnHCl] > 1.0 M and is complete at [GdnHCl] > or = 6.0 M. Upon unfolding, two fluorescence peaks were observed. One peak was assigned to the fluorescence of Trp-214 in a polar environment, and the other peak was assigned to tyrosine fluorescence. A reduction of the fluorescence intensity of the two peaks upon binding of the probes to the denatured HSA indicates that Tyr-263 in subdomain IIA is one of the tyrosine residues responsible for the second fluorescence peak. The results were confirmed by measuring the fluorescence spectra and lifetimes of denatured HSA at different excitation wavelengths, and of L-tryptophan and L-tyrosine free in buffer. The measured lifetimes of denatured HSA are typical of tryptophan in a polar environment and are slightly reduced upon probe binding. Dilution of the denatured HSA by buffer results in a partial refolding of subdomain IIA. This partial refolding is attributed to some swelling of the binding site caused by water. The swelling prevents a full recovery from the denatured state.
通过检测人血清白蛋白（HSA）在天然、去折叠和重折叠状态下荧光的变化，对其亚结构域IIA结合位点进行了表征。该研究在不存在和存在小分子探针的情况下进行，采用稳态和时间分辨荧光测量。2-吡啶酮、3-吡啶酮和4-吡啶酮具有与许多药物中发现的分子结构相似的结构，在此用作探针。发现它们特异性结合在亚结构域IIA中，并导致天然HSA中位于同一亚结构域的Trp-214残基的荧光强度和寿命降低。发现从Trp-214荧光到探针的能量转移效率取决于供体荧光与受体吸收之间的光谱重叠程度。在探针结合到亚结构域IIA后，使用福斯特理论计算供体和受体之间的距离。计算得到的猝灭速率常数和结合常数也显示取决于光谱重叠程度。结果表明复合物中存在静态猝灭机制。在盐酸胍（GdnHCl）存在下，HSA的变性在[GdnHCl] > 1.0 M时开始，在[GdnHCl] >或 = 6.0 M时完成。去折叠时，观察到两个荧光峰。一个峰归属于极性环境中Trp-214的荧光，另一个峰归属于酪氨酸荧光。探针与变性HSA结合后两个峰的荧光强度降低表明亚结构域IIA中的Tyr-263是负责第二个荧光峰的酪氨酸残基之一。通过测量不同激发波长下变性HSA以及缓冲液中游离的L-色氨酸和L-酪氨酸的荧光光谱和寿命，证实了该结果。测得的变性HSA的寿命是极性环境中色氨酸的典型寿命，并且在探针结合后略有降低。用缓冲液稀释变性HSA会导致亚结构域IIA部分重折叠。这种部分重折叠归因于水引起的结合位点的一些膨胀。这种膨胀阻止了从变性状态的完全恢复。
