Khan M M, Muzammil S, Tayyab S
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, India.
Biochimie. 2000 Mar;82(3):203-9. doi: 10.1016/s0300-9084(00)00205-4.
Chloroform-induced conformational changes of bilirubin (BR) bound to different serum albumins were studied by circular dichroism (CD) and fluorescence spectroscopy. Addition of a small amount of chloroform ( approximately 20 mM) to a solution containing 20 microM albumin and 15 microM BR changed the sign order and magnitude of the characteristic CD spectra of all BR-albumin complexes except BR-PSA complex which showed abnormal behavior. Monosignate negative CD Cotton effects (CDCEs) of BR complexed with SSA, GSA and BuSA were transformed into bisignate CDCEs in presence of chloroform akin to those exhibited by chloroform free solution of BR-HSA complex, indicating that the pigment acquired right handed plus (P) chirality when chloroform was added to these complexes. Bisignate CD spectra of BR complexed with HSA and BSA showed complete inversion upon addition of chloroform corroborating earlier findings. On the other hand, changes observed with BR-RSA complex were slightly different showing an additional CD band of weak intensity centered around 390 nm though inversion of CDCEs was similar to that of BR-HSA complex. Monosignate CD spectra of BR-PSA complex also showed three CD bands occurring at 409, 470 and 514 nm after chloroform addition. These results indicated significant but different effects of chloroform on the conformation of bound BR in BR-albumin complexes which can be ascribed to the changes in the exciton chirality of bilirubin probably due to altered hydrophobic microenvironment induced by the binding of chloroform at or near the ligand binding site. Chloroform severely quenched the intrinsic tryptophan fluorescence of the protein and shifted the emission maxima towards blue region in all the albumins except PSA. However, quantitative differences in both quenching and blue shift were noted in different serum albumins. This suggests that chloroform probably binds in the close vicinity of tryptophan residue(s) located in subdomain(s) IIA or IB and II both. The fluorescence of BR-albumin complexes was also found to be sensitive to the presence of a small amount of chloroform. But the changes observed in the fluorescence of the bound pigment in presence of chloroform were less marked as compared to the changes in the intrinsic fluorescence of protein per se. Taken together, these results suggest that there is at least one conserved site for chloroform binding in all these albumins which is at or near the BR binding site.
通过圆二色性（CD）和荧光光谱研究了氯仿诱导的与不同血清白蛋白结合的胆红素（BR）的构象变化。向含有20μM白蛋白和15μM BR的溶液中加入少量氯仿（约20 mM），除了表现出异常行为的BR-PSA复合物外，所有BR-白蛋白复合物的特征CD光谱的符号顺序和大小都发生了变化。与SSA、GSA和BuSA络合的BR的单峰负CD Cotton效应（CDCEs）在氯仿存在下转变为双峰CDCEs，类似于BR-HSA复合物的无氯仿溶液所表现出的情况，这表明当向这些复合物中加入氯仿时，该色素获得了右手正（P）手性。与HSA和BSA络合的BR的双峰CD光谱在加入氯仿后显示出完全反转，证实了早期的发现。另一方面，BR-RSA复合物观察到的变化略有不同，尽管CDCEs的反转与BR-HSA复合物相似，但在390 nm左右出现了一个强度较弱的额外CD带。氯仿加入后，BR-PSA复合物的单峰CD光谱在409、470和514 nm处也出现了三个CD带。这些结果表明氯仿对BR-白蛋白复合物中结合的BR的构象有显著但不同的影响，这可能归因于胆红素激子手性的变化，可能是由于氯仿在配体结合位点或其附近结合引起的疏水微环境改变。氯仿严重淬灭了蛋白质的内在色氨酸荧光，并使除PSA外的所有白蛋白的发射最大值向蓝色区域移动。然而，在不同血清白蛋白中观察到淬灭和蓝移的定量差异。这表明氯仿可能在位于IIA或IB亚结构域以及II两者中的色氨酸残基附近结合。还发现BR-白蛋白复合物的荧光对少量氯仿的存在敏感。但是，与蛋白质本身的内在荧光变化相比，在氯仿存在下结合色素的荧光变化不太明显。综上所述，这些结果表明在所有这些白蛋白中至少有一个氯仿结合的保守位点，它在BR结合位点或其附近。
