Vermishian I G, Sharoian S G, Antonian A A, Grigorian N A, Mardanian S S, Khoetsian A V, Markarian Sh A
Biofizika. 2008 Mar-Apr;53(2):213-21.
The effect of inhibitors, 1-deazaadenosine (1-dAdo) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), on the conformation of adenosine deaminase was studied using the method of selective quenching of fluorescence emission by acrylamide, I- and Cs+. Both in free adenosine deaminase and in its complexes with the inhibitors, the wavelength maxima and half-width of the emission characterize the environment of fluorescing tryptophan residues in adenosine deaminase as weak polar with limited access to solvent. The formation of complexes with the ground state inhibitors used did not quench or change the main emission characteristics of tryptophan fluorescence in adenosine deaminase. Small blue shifts of emission maxima were observed upon quenching in all three samples. The Stern-Volmer parameters of tryptophan fluorescence quenching by acrylamide were not essentially influenced by complex formation of the enzyme with the inhibitors: in general, the folding of the enzyme molecule in the complexes is not perturbed. On the contrary, the emission quenching by charged heavy ions, I- and Cs+, in the complexes was hindered in comparison with free adenosine deaminase. In the complex with 1-deazaadenosine, the parameters for quenching by both ions evidence the essential worsening of their interaction with tryptophans. In the complex with erythro-9-(2-hydroxy-3-nonyl)adenine, along with the worse quenching by I-, complete prohibition of quenching by Cs+ was observed. These data indicate that the local environments of fluorescing tryptophan residues is substantially distorted compared with free adenosine deaminase, which leads to their screening from charged heavy ions.
使用丙烯酰胺、碘离子（I⁻）和铯离子（Cs⁺）选择性猝灭荧光发射的方法，研究了抑制剂1-脱氮腺苷（1-dAdo）和赤型-9-（2-羟基-3-壬基）腺嘌呤（EHNA）对腺苷脱氨酶构象的影响。无论是在游离的腺苷脱氨酶中，还是在其与抑制剂的复合物中，发射光的波长最大值和半高宽表明，腺苷脱氨酶中发荧光的色氨酸残基所处环境为弱极性，与溶剂的接触有限。与所用的基态抑制剂形成复合物，并不会猝灭或改变腺苷脱氨酶中色氨酸荧光的主要发射特性。在所有三个样品中猝灭时，均观察到发射最大值有小的蓝移。丙烯酰胺对色氨酸荧光猝灭的斯特恩-沃尔默参数，基本上不受酶与抑制剂形成复合物的影响：总体而言，复合物中酶分子的折叠未受干扰。相反，与游离的腺苷脱氨酶相比，复合物中带电荷的重离子I⁻和Cs⁺对发射的猝灭受到阻碍。在与1-脱氮腺苷形成的复合物中，两种离子的猝灭参数表明它们与色氨酸的相互作用明显变差。在与赤型-9-（2-羟基-3-壬基）腺嘌呤形成的复合物中，除了I⁻的猝灭变差外，还观察到Cs⁺完全禁止猝灭。这些数据表明，与游离的腺苷脱氨酶相比，发荧光的色氨酸残基的局部环境发生了实质性扭曲，这导致它们被带电荷的重离子屏蔽。
