Kamal J K Amisha, Behere Digambar V
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India.
Indian J Biochem Biophys. 2005 Feb;42(1):7-12.
The binding of monomeric heme to human serum albumin (HSA) was investigated using steady-state fluorescence, circular dichroism (CD) and optical difference spectroscopic (ODS) techniques. The existence of one strong binding site for heme on HSA was confirmed by titrating heme with HSA and following the quenching of tryptophan (Trp214) fluorescence emission intensity that occurred due to energy transfer. Up to around 1:1 stoichiometric ratio of HSA/heme, the quenching was observed to be very strong, however at higher ratios the quenching progressed very weakly. Similarly, the negative CD band centered at -397 nm, which appeared on adding heme to HSA, increased in intensity on sequential addition of heme up to [heme]/[HSA] = 1. Titration of HSA with heme was followed by ODS and the dissociation constant K(D) = (4.0 +/- 1.0) x 10(-5) M was deduced. Results have been explained on the basis of Michaelis-Menton type of mechanism for the heme binding, in which heme first binds reversibly to His146 at the surface of the protein to form an intermediate complex, followed by irreversible binding to Tyr161 in the interior of the protein.
采用稳态荧光、圆二色性（CD）和光学差示光谱（ODS）技术研究了单体血红素与人血清白蛋白（HSA）的结合。通过用HSA滴定血红素并跟踪由于能量转移导致的色氨酸（Trp214）荧光发射强度的猝灭，证实了HSA上存在一个血红素强结合位点。在HSA/血红素化学计量比约为1:1之前，观察到猝灭非常强烈，但在更高比例时，猝灭进展非常微弱。同样，在向HSA中添加血红素时出现的以-397 nm为中心的负CD带，在依次添加血红素直至[血红素]/[HSA]=1时强度增加。用ODS跟踪HSA与血红素的滴定，并推导出解离常数K(D)=(4.0±1.0)×10(-5)M。基于血红素结合的米氏-门坦类型机制对结果进行了解释，其中血红素首先与蛋白质表面的His146可逆结合形成中间复合物，随后与蛋白质内部的Tyr161不可逆结合。
