warfarin and prodan

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.

Topics: 2-Naphthylamine; Binding Sites; Binding, Competitive; Cysteine; Energy Transfer; Fluorescent Dyes; Hot Temperature; Humans; Palmitic Acid; Protein Conformation; Protein Denaturation; Protein Folding; Serum Albumin; Spectrometry, Fluorescence; Tryptophan; Warfarin

The fluorescent probe Prodan (6-propionyl-2-dimethyl-aminonaphthalene) binds with high affinity to human serum albumin (HSA). The spectral characteristics of the Prodan bound to the protein are very different from the free Prodan in solution. These differences allowed the spectra to be deconvoluted into log-normal bands in order to quantify the bound and unbound ligand and to calculate the binding constant at different temperatures. From such temperature dependence, we found the binding to be exothermic with a van't Hoff enthalpy of -22.8 kJ mol-1. Thermodynamic analysis suggests that the interaction may be mainly caused by hydrophobic forces and electrostatic interactions. The above analysis of the spectra and the measures of the fluorescence polarization during the successive presence of six specific drugs suggest that the Prodan binding site corresponds with the warfarin binding site on HSA, whereas under the present experimental conditions the other characteristic binding sites of HSA were not affected. Thus, this fluorescent probe provides a rapid and simple means for the characterization of a specific binding site on HSA and also for detecting potential or nonspecific drug-protein interactions.

Topics: 2-Naphthylamine; Binding Sites; Circular Dichroism; Fluorescence Polarization; Fluorescent Dyes; Humans; In Vitro Techniques; Protein Binding; Serum Albumin; Warfarin