Phenylbutazone

Phenylbutazone is known to have affinity for Human Serum Albumin (HSA) and to bind (interact) to drug binding site I on HSA. Those were confirmed using our phenylbutazone with Surface Plasmon Resonance (SPR) and a method using fluorescent probes. 【SPR】 Dose responses of phenylbutazone to HSA were confirmed by SPR. Biacore, as a SPR biosensor, was used for the assay, according to the user’s guide of the instrument.

＜Assay condition＞ Sensor Chip: Series S Sensor Chip CM5, Immobilization: HAS (Amine Coupling method), Buffer : 5%DMSO in PBS. ＜Result＞ “Square wave” sensorgrams were exhibited at each concentration, and concentration dependent binding of phenylbutazone to HSA was confirmed. 【Method using fluorescent probes】The drug biding site of phenylbutazone was confirmed using fluorescent probes which bind to drug binding site on HSA. Dansylamide (DNSA) [D5405] was used as fluorescent probe for site I, and dansylglycine (DNSG) [D5406], BD140 [D4898] were used as fluorescent probes for site II, and then bindings to site I and site II were confirmed.

＜Assay condition＞ Buffer: 1 % DMSO in phosphate buffer (pH 7.2 - 7.5); HSA: 5 µM (DNSA), 20 µM (DNSG, BD140) (50 µL/well) (Fatty acid free HSA is recommended.); Phenylbutazone: each concentration (50 µL/well); DNSA: 80 µM, DNSG: 20 µM, BD140: 20 µM (50 µL/well); Incubation: 20-25 °C for 30 min; Measurement: plate-reader with excitation = 365 nm and emission = 480 nm (DNSA, DNSG), with excitation = 365 nm and emission = 585 nm (BD140). ＜Result＞ As shown in upper diagram, inhibition against binding of dansylamide which is fluorescent probe for site I by phenylbutazone was confirmed. And also, little or extremely weak inhibition against binding of dansylglycine and BD140 which are fluorescent probes for site II was confirmed. In these ways, our phenylbutazone can be used for study of interaction with HSA. Also, DNSA [D5405], DNSG [D5406] and BD140 [D4898] can be used for study of drug binding site on HSA.