Human Serum Albumin (HSA) binds to a wide variety of drugs at two primary binding sites (I: blue and II: yellow), and can have a significant impact on their pharmacokinetics and pharmacological effects.
• Detectable drug binding to HSA at site I and II.
• Simple and quick assay can be performed.
The fluorescent probes dansylamide [D5405] and dansylglycine [D5406] selectively interact with sites I and II, respectively.1,2) BD140 [D4898] also selectively binds to drug binding site II.3) Changes in probe fluorescence are analyzed by fluorescence titrations as a result of competitive displacement by drugs and pharmaceuticals. The pattern of fluorescent probe displacement by these reagents enabled the identification of two specific drug binding sites, which illustrates their usefulness for the characterization of the binding sites in HSA.
[Reagents] • 1% DMSO in 10mM phosphate buffer (pH 7.2-7.5) • Human Serum Albumin (HSA) (fatty acid free) • Various sample compounds • HSA binding fluorescent probe : dansylamide [D5405], dansylglycine [D5406], BD140 [D4898]
[Procedure] (1) Prepare HSA, each fluorescent probe, and various sample compounds. (2) Apply each solution to black 96-well plate for fluorescent measurement. (Ex. 50µL each) (3) Stir gently and let stand for 30 minutes with shading at room temperature (20-25°C). (4) Measure the fluorescence with a plate reader.
* As positive control, warfarin is often used for drug binding site I and ibuprofen is often used for drug binding site II. * Detergents sometimes effects the reaction. Consideration of the application conditions are recommended. * For the application of dansylglycine and BD140, fatty acid free HSA is recommended for use. (Please refer to data in the below brochure.)
[Assay condition] Buffer: 1% DMSO in phosphate buffer (pH7.2-7.5), HSA (fatty acid free): 5 µM (50µL/well), Drugs: each concentration (50 µL/well), Each fluorescent probe: 80 µM (50 µL/well), Incubation: 20-25°C for 30 min, Measurement: plate-reader, excitation=365 nm, emission=480 nm (Dansylamide and Dansylglycine) or 585nm (BD140).
[Our testing results]
• The binding of dansylamide [D5405] to HSA (fatty acid free) Inhibition was achieved by warfarin, phenylbutazone and triiodobenzoic acid bound to drug binding site I. Alternatively, it was not inhibited by ibuprofen and flurbiprofen bound to drug binding site II, completely or partially. • Binding of dansylglycine [D5406] and BD140 [D4898] to HSA (fatty acid free) It was not partially inhibited by warfarin and phenylbutazone bound to drug binding site I. However, it was inhibited by ibuprofen, flurbiprofen and triiodzendoic acid bound to drug binding site II.
Dansylamide makes it possible to confirm whether a drug binds to drug binding site I on HSA. Dansylglycine and BD140 makes it possible to confirm whether the drug binds to drug binding site II on HSA.