title: Binding of naproxen enantiomers to human serum albumin studied by fluorescence and room-temperature phosphorescence.
abstract: The interaction of the enantiomers of the non-steroidal anti-inflammatory drug naproxen (NPX) with human serum albumin (HSA) has been investigated using fluorescence and phosphorescence spectroscopy in the steady-state and time-resolved mode. The absorption, fluorescence excitation, and fluorescence emission spectra of (S)-NPX and (R)-NPX differ in shape in the presence of HSA, indicating that these enantiomers experience a different environment when bound. In solutions containing 0.2M KI, complexation with HSA results in a strongly increased NPX fluorescence intensity and a decreased NPX phosphorescence intensity due to the inhibition of the collisional interaction with the heavy atom iodide. Fluorescence intensity curves obtained upon selective excitation of NPX show 8-fold different slopes for bound and free NPX. No significant difference in the binding constants of (3.8±0.6)×10(5) M(-1) for (S)-NPX and (3.9±0.6)×10(5) M(-1) for (R)-NPX was found. Furthermore, the addition of NPX quenches the phosphorescence of the single tryptophan in HSA (Trp-214) based on Dexter energy transfer. The short-range nature of this mechanism explains the upward curvature of the Stern-Volmer plot observed for HSA: At low concentrations NPX binds to HSA at a distance from Trp-214 and no quenching occurs, whereas at high NPX concentrations the phosphorescence intensity decreases due to dynamic quenching by NPX diffusing into site I from the bulk solution. The dynamic quenching observed in the Stern-Volmer plots based on the longest phosphorescence lifetime indicates an overall binding constant to HSA of about 3×10(5) M(-1) for both enantiomers.
authors.affiliation: Biomolecular Analysis and Spectroscopy, LaserLaB, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Ivonne
authors.initials: I
authors.lastname: Lammers
authors.forename: Virginie
authors.initials: V
authors.lastname: Lhiaubet-Vallet
authors.forename: Freek
authors.initials: F
authors.lastname: Ariese
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Miranda
authors.forename: Cees
authors.initials: C
authors.lastname: Gooijer
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D008163:Luminescent Measurements; D009288:Naproxen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D012709:Serum Albumin; D009288:Naproxen