Binding of naproxen enantiomers to human serum albumin studied by fluorescence and room-temperature phosphorescence

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 0.2 M 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) × 105 M−1 for (S)-NPX and (3.9 ± 0.6) × 105 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 × 105 M−1 for both enantiomers.

Graphical abstractStatic and dynamic interactions of HSA with the drugs (S)- or (R)-naproxen studied with phosphorescence and fluorescence.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Fluorescence spectra of (S)- and (R)-naproxen show enantioselective binding to HSA. ► Dynamics were studied at ns (fluorescence) to ms (phosphorescence) timescales. ► (S)- and (R)-naproxen bound to HSA are effectively shielded from iodide quenching. ► Phosphorescence of HSA showed 1:1 binding, followed by dynamic quenching.