Biophysical Characterization of Interactions Between Human Salivary Proteins and Quinine

Saliva is the first biological medium encountered by bitter compounds in the oral cavity, and salivary proteins have been indirectly implicated in bitterness perception. However, direct biophysical characterization of salivary protein-bitterant interactions has not been reported. This study investigated interactions between salivary proteins and the model bitterant quinine using complementary biophysical techniques. Pooled whole saliva (WS) from 44 healthy volunteers was separated into high- (HMW) and low-molecular-weight (LMW) fractions using a 3 kDa filter. Protein-quinine interactions were characterized by fluorescence spectroscopy, sedimentation velocity analytical ultracentrifugation (SV-AUC), and isothermal titration calorimetry (ITC). Fluorescence spectroscopy showed a concentration-dependent reduction in the apparent unbound quinine, reaching 59.28 ± 0.89% at 0.6 mg/mL protein in WS, with greater apparent binding in the LMW fraction than the HMW fraction. SV-AUC provided evidence of protein-quinine complex formation, with the LMW fraction exhibiting the clearest changes through the appearance of new, higher sedimentation coefficient species, suggesting quinine-induced multimerization. ITC revealed endothermic binding across all three saliva systems, with the LMW fraction exhibiting the greatest change in apparent binding enthalpy (∆H = 1.053 ± 0.009 kcal/ mol) compared with WS (∆H = 0.321 ± 0.548 kcal/ mol) and HMW fraction (∆H = 0.081 ± 0.158 kcal/ mol). These findings converge to demonstrate that human salivary proteins interact with quinine, with the LMW species (< 3 kDa) exhibiting the strongest apparent binding (17.53 ± 1.58% reduction in unbound quinine at 0.2 mg/mL protein from the fluorescence measurements). This work provides the first biophysical characterization of salivary protein-bitterant interactions and establishes a foundation for future investigations into the role of salivary proteins in bitterness perception. Graphical Abstract