Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

The genetic predisposition to taste 6-n-propylthiouracil (PROP) varies among individuals and is associated with salivary levels of Ps-1 and II-2 peptides, belonging to the basic proline-rich protein family (bPRP). We evaluated the role of these proteins and free amino acids that selectively interact with the PROP molecule, in modulating bitter taste responsiveness. Subjects were classified by their PROP taster status based on ratings of perceived taste intensity for PROP and NaCl solutions. Quantitative and qualitative determinations of Ps-1 and II-2 proteins in unstimulated saliva were performed by HPLC-ESI-MS analysis. Subjects rated PROP bitterness after supplementation with Ps-1 and II-2, and two amino acids (L-Arg and L-Lys) whose interaction with PROP was demonstrated by (1)H-NMR spectroscopy. ANOVA showed that salivary levels of II-2 and Ps-1 proteins were higher in unstimulated saliva of PROP super-tasters and medium tasters than in non-tasters. Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter taste responsiveness, and this effect was specific to the non-taster group.(1)H-NMR results showed that the interaction between PROP and L-Arg is stronger than that involving L-Lys, and taste experiments confirmed that oral supplementation with these two amino acids increased PROP bitterness intensity, more for L-Arg than for L-Lys. These data suggest that Ps-1 protein facilitates PROP bitter taste perception and identifies a role for free L-Arg and L-Lys in PROP tasting.

Thiourea tasting can be predictive of individual differences in bitter taste responses, general food preferences and eating behavior, and could be correlated with saliva chemical composition. We investigated the possible relationship between PROP bitter taste responsiveness and the salivary proteome in subjects genotyped for TAS2R38 and gustin gene polymorphisms. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 25±3 y) to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the 'taster' variant (PAV haplotype) of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and nontasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in nontaster un-stimulated saliva, and that PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait.