Fluorescence spectroscopic study of serum albumin-bromadiolone interaction: fluorimetric determination of bromadiolone.
Bromadiolone (BRD), a substituted 4-hydroxycoumarin derivative, is known to possess anti-coagulant activity with acute toxicity. In this paper, we report a study on the interaction of bromadiolone with the plasma proteins bovine serum albumin (BSA) and human serum albumin (HSA), using the intrinsic fluorescence emission properties of bromadiolone. Bromadiolone is weakly fluorescent in aqueous buffer medium, with an emission at 397 nm. Binding of bromadiolone with serum albumins (SA) leads to a marked enhancement in the fluorescence emission intensity and steady state fluorescence anisotropy (r(ss)), accompanied by a blueshift of 10 nm. In the serum albumin-bromadiolone complex, selective excitation of tryptophan (Trp) residue results in emission from bromadiolone, thereby indicating a Förster type energy transfer from Trp to BRD. This quenching of Trp fluorescence by BRD was used to estimate the binding constant of the SA-BRD complex. The binding constants for BRD with BSA and HSA were 7.5 x 10(4) and 3.7 x 10(5)L mol(-1), respectively. Based on this, a new method involving SA as fluorescence-enhancing reagent for estimation of BRD in aqueous samples has been suggested. The detection limits of bromadiolone under the optimum conditions were 0.77 and 0.19 microg mL(-1) in presence of BSA and HSA, respectively.
Deepa
Subbiah
S
Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, India.
Mishra
Ashok K
AK
D015110:4-Hydroxycoumarins / Q000032:analysis* / Q000737:chemistry; D000465:Algorithms; D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D011485:Protein Binding; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
D015110:4-Hydroxycoumarins; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; C021636:bromadiolone