Johnson J D, El-Bayoumi M A, Weber L D, Tulinsky A
Biochemistry. 1979 Apr 3;18(7):1292-6. doi: 10.1021/bi00574a027.
The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.
荧光探针1-苯胺基萘-8-磺酸盐（Ans）在pH 3.6时与α-胰凝乳蛋白酶（α-CHT）结合，伴随Ans荧光显著增强且发射峰最大值向较短波长移动。我们的研究表明，一个Ans分子在与α-CHT的活性位点或2-对甲苯胺基萘-6-磺酸盐结合位点不同的位点与α-CHT结合。Ans在pH 3.6和6.4时的结合常数大致相同（10⁴ M⁻¹）。纳秒荧光去极化数据表明Ans与α-CHT紧密结合。低pH时Ans与α-CHT结合导致的荧光增强可能是由于其与疏水位点结合，或者是与在Ans激发态寿命期间局部偶极不弛豫的位点结合。随着pH升高，Ans-α-CHT复合物的荧光强度明显降低；发射峰最大值向较长波长移动。荧光衰减曲线对pH表现出相应的敏感性。pH对Ans-α-CHT荧光的影响可以用极性残基和Ans结合位点处水分子移动程度不同的α-CHT构象异构体之间的pH依赖性平衡，或者Ans结合位点的结构变化来解释。
