| Summary: | 碩士 === 國立臺灣大學 === 化學學系 === 83 ===
As a carrier protein, serum albumin binds with a wide variety of small molecules, e. g., amino acids, metabolites, and drugs. The UV - visible absorption and fluorescence spectral techniques have frequently been used to study the binding interactions between small ligands and proteins. These techniques, when used properly, can provide valuable information regarding stoichiometry, mechanism, and binding constant. For ligands with high affinity constants (Ka = 105 M - 1 or greater) the fluorescence binding technique is indispensable. Determination of the bindig constant of protein with an intrinsic fluorescent probe is quite simple when using the nonlinear curve - fitting (two - parameter) technique.
In this paper, the interactions of several fluorescent ligands, including warfarin, bromophenol (BPB), MC 540, salicylate, 1 - anilino - 8 - naphthalenesulfonic acid (1, 8 - ANS), and dansylglycina with bovine or human serum albumia (BSA) have been studied by using the absorption and fluorescence spectral techniques. The fluorescence (max. at 630 nm) of BPB increased ca. 20 - fold upon binding on BSA. The binding constant has been determined to be ca. 1.6 (± 0.03) × 106 M-1. Deoxycholate (binding on the Ⅲ A site) induced conformational changes on BSA that influenced binding of other ligands to their binding sites on BSA. The addition of deoxycholate to the BPB - BSA complex caused a biphasic fluorescence changes; an additional 4 - fold fluorescence enhancement was observed initially, but as [deoxycholate] increased (forming dimer or micelles) the fluorescene of BPB decreased. Deoxycholate binding also caused a blue - shife (15 nm) on the emission maximum of BSA, and the two tryptophan residues of BSA became inaccessible to quencher iodide ion. A similar biphasic effect of deoxycholate on the fluorescence behavior of warfarin and MC 540 was also observed. Mechanisms for the binding interactions between small ligands and albumin and the influence by deoxycholate micelles have been proposed.
The increases in fluorescence emission upon complex formation were used to determine the binding constants using the nonlinear curvefitting method. The binding constants of MC - 540 for warfarin and dansylglycine were determined to be 1.28 (± 0.07) × 106、3.51 (± 0.03) × 105 and 1.61 (± 0.04) × 105 M-1, respectively. The fluorescence displacement studies suggest that the primary binding site of MC 540 is located in the subdomain Ⅲ A of HSA. On the other hand, dansylglycine may share a common binding region with phenylbutazone. The present study has also demonstrated that the binding site for warfarin is different from that for dansylglycine, and when the former site is occupied by warfarin, the binding of dansylglycine to HSA is also inhibited. In conclusion, the present research provide some evidence that some binding sites on serum albumin are mutually interacting to each other when bound with specific ligands.
|
|---|
