Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method.
The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants (K(a)) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02x10(7) and 2.07x10(4)L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S(0)-->S(1) transition of esculin (lambda(ex)(max) approximately 340nm) appears, which is similar to the lambda(em)(max) of BSA and HSA. The critical distance (R(0)) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.
Institute of Food Engineering, College of Life & Environment Science, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, PR China. jianboxiao@yahoo.com
Jianbo
J
Xiao
Xinlin
X
Wei
Yuanfeng
Y
Wang
Chunxi
C
Liu
D000818:Animals; D002417:Cattle; D004929:Esculin / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D012709:Serum Albumin; D004929:Esculin; D012710:Serum Albumin, Bovine