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Title: Binding of heme to human serum albumin: Steady-state fluorescence, circular dichroism and optical difference spectroscopic studies
Authors: Kamal, J K Amisha
Behere, Digambar V
Keywords: human serum albumin (HSA)
methemalbumin (MHA)
steady-state fluorescence
circular dichroism
optical difference spectroscopy
two stage binding
Issue Date: Feb-2005
Publisher: CSIR
IPC CodeG01J 3/00
Abstract: 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 KD = (4.0±1.0)×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
Page(s): 7-12
ISSN: 0301-1208
Source:IJBB Vol.42(1) [February 2005]

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