Department of Biology, Faculty of Science, Razi University, 67149-67346 Kermanshah, Iran.
J Photochem Photobiol B. 2009 Dec 2;97(3):161-8. doi: 10.1016/j.jphotobiol.2009.09.005. Epub 2009 Sep 19.
The effect of celecoxib, a sulfonamide drug, on the structure and function of human carbonic anhydrase II (hCA II) was investigated by various spectroscopic techniques such as UV-Vis, fluorescence and circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC), in 20 mM Tris, pH 7.75 at 27 degrees C. Kinetic results revealed that celecoxib inhibits the esterase activity of hCA II in a linear competitive manner with K(i) = 61.61 + or -3.05 nM. DSC data indicated that the thermal stability of the enzyme has a minor increment in the presence of celecoxib. Fluorescence measurements showed that the celecoxib acts as a quencher of the enzyme fluorescence and calculation of the protein surface hydrophobicity (PSH), using 1-anilinonaphthalene-8-sulfonate (ANS), revealed the decrement of its PSH upon interaction with the drug. Acrylamide quenching experiments indicated the less accessibility of the tryptophan residues to the acrylamide due to the presence of celecoxib. Stern-Volmer analysis of quenching data at different temperatures elucidated that the quenching of intrinsic fluorescence of hCA II is occurred through a static quenching mechanism. Analysis of the thermodynamic parameters of binding showed that hydrogen bonding and hydrophobic interactions play the major role in stabilization of the enzyme-drug complex. The Job's plot confirmed the existence of one binding site for celecoxib in hCA II. The far- and near-UV CD experiments indicated that celecoxib causes a little increment in alpha-helicity content of hCA II whereas its flexibility is decreased somewhat upon celecoxib binding.
在 27°C、20mM Tris、pH7.75 的条件下，采用紫外可见光谱、荧光光谱和圆二色性（CD）光谱以及差示扫描量热法（DSC）等多种光谱技术研究了磺胺类药物塞来昔布对人碳酸酐酶 II（hCA II）结构和功能的影响。动力学结果表明，塞来昔布以线性竞争方式抑制 hCA II 的酯酶活性，K(i)值为 61.61±3.05nM。DSC 数据表明，酶的热稳定性在存在塞来昔布时有轻微增加。荧光测量表明，塞来昔布是酶荧光的猝灭剂，使用 1-苯胺基萘-8-磺酸盐（ANS）计算蛋白质表面疏水性（PSH）表明，药物相互作用后其 PSH 减少。丙烯酰胺猝灭实验表明，由于存在塞来昔布，色氨酸残基对丙烯酰胺的可及性降低。在不同温度下的猝灭数据的 Stern-Volmer 分析表明，hCA II 内源性荧光的猝灭是通过静态猝灭机制发生的。结合热力学参数分析表明，氢键和疏水相互作用在稳定酶-药物复合物中起主要作用。Job 的图谱证实了塞来昔布在 hCA II 中存在一个结合位点。远紫外和近紫外 CD 实验表明，塞来昔布导致 hCA II 的α-螺旋含量略有增加，而结合塞来昔布后其柔韧性略有降低。
