Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC-High-Resolution ESI-MS/MS.

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.

Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease.

Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser22 (Phos) → Phe variant.

During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ∼22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22 (phos)→Phe.

Aggregation of the salivary proline-rich protein IB5 in the presence of the tannin EgCG.

In the mouth, proline-rich proteins (PRP), which are major components of stimulated saliva, interact with tannins contained in food. We report in vitro interactions of the tannin epigallocatechin gallate (EgCG), with a basic salivary PRP, IB5, studied through electrospray ionization mass spectrometry (ESI-MS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS). In dilute protein (IB5) solutions of low ionic strength (1 mM), the proteins repel each other, and the tannins bind to nonaggregated proteins. ESI-MS experiments determine the populations of nonaggregated proteins that have bound various numbers of tannin molecules. These populations match approximately the Poisson distribution for binding to n = 8 sites on the protein. MS/MS experiments confirm that complexes containing n = 1 to 8 EgCG molecules are dissociated with the same energy. Assuming that the 8 sites are equivalent, we calculate a binding isotherm, with a binding free energy Δµ = 7.26RT(a) (K(d) = 706 µM). In protein solutions that are more concentrated (0.21 mM) and at higher ionic strength (50 mM, pH 5.5), the tannins can bridge the proteins together. DLS experiments measure the number of proteins per aggregate. This number rises rapidly when the EgCG concentration exceeds a threshold (0.2 mM EgCG for 0.21 mM of IB5). SAXS experiments indicate that the aggregates have a core-corona structure. The core contains proteins that have bound at least 3 tannins and the corona has proteins with fewer bound tannins. These aggregates coexist with nonaggregated proteins. Increasing the tannin concentration beyond the threshold causes the transfer of proteins to the aggregates and a fast rise of the number of proteins per aggregate. A poisoned growth model explains this fast rise. Very large cationic aggregates, containing up to 10,000 proteins, are formed at tannin concentrations (2 mM) slightly above the aggregation threshold (0.2 mM).

The colloidal state of tannins impacts the nature of their interaction with proteins: the case of salivary proline-rich protein/procyanidins binding.

While the definition of tannins has been historically associated with its propensity to bind proteins in a nonspecific way, it is now admitted that specific interaction also occurs. The case of the astringency perception is a good example to illustrate this phenomenon: astringency is commonly described as a tactile sensation induced by the precipitation of a complex composed of proline-rich proteins present in the human saliva and tannins present in beverages such as tea or red wines. In the present work, the interactions between a human saliva protein segment and three different procyanidins (B1, B3, and C2) were investigated at the atomic level by NMR and molecular dynamics. The data provided evidence for (i) an increase in affinity compared to shortest human saliva peptides, which is accounted for by protein "wraping around" the tannin, (ii) a specificity in the interaction below tannin critical micelle concentration (CMC) of ca. 10 mM, with an affinity scale such that C2 > B1 > B3, and (iii) a nonspecific binding above tannin CMC that conducts irremediably to the precipitation of the tannins/protein complex. Such physicochemical findings describe in accurate terms saliva protein-tannin interactions and provide support for a more subtle description by oenologists of wine astringency perception in the mouth.

First identification of tannin-binding proteins in saliva of Papio hamadryas using MS/MS mass spectrometry.

Hamadryas baboons possess salivary proline-rich proteins (PRP), as indicated by the presence of pink-staining protein bands using 1D SDS gel electrophoresis and Coomassie R250 staining. The ability of these protein bands to interact with tannic acid was further examined. In a tannin-binding assay using 5 µg tannic acid mixed with hamadryas whole saliva, we recently found four distinct protein bands of apparently 72, 55, 20, and 15 kDa that were precipitated during the experiments. In this work, we were able to identify these protein bands in a follow-up analysis using MS/MS mass spectrometry after excising such bands out of air-dried gels. Albumin and α-amylase were present in the tannic acid-protein complexes, with albumin already known to nonspecifically interact with a great diversity of chemical compounds. More interesting, we also identified a basic PRP and a cystatin precursor protein. This was the first successful attempt to identify a PRP from precipitated tannin-protein complexes in hamadryas baboons using MS/MS mass spectrometry. On the other hand, the role of cystatins in tannin binding is not yet well understood. However, there are recent reports on cystatin expression in saliva of rats responding to astringent dietary compounds. In conclusion, the follow-up data on tannin-binding proteins present in salivary secretions from hamadryas baboons adds important knowledge to primate physiology and feeding ecology, in order to shed light on the establishment and development of food adaptations in primates. It also demonstrates that tannin binding is characteristic for PRP, but might not be restricted to this particular group of proteins in primate species.

Proline-rich salivary proteins have extended conformations.

Three basic proline-rich salivary proteins have been produced through the recombinant route. IB5 is a small basic proline-rich protein that is involved in the binding of plant tannins in the oral cavity. II-1 is a larger protein with a closely related backbone; it is glycosylated, and it is also able to bind plant tannins. II-1 ng has the same polypeptidic backbone as II-1, but it is not glycosylated. Small angle x-ray scattering experiments on dilute solutions of these proteins confirm that they are intrinsically disordered. IB5 and II-1 ng can be described through a chain model including a persistence length and cross section. The measured radii of gyration (Rg=27.9 and 41.0+/-1 A respectively) and largest distances (rmax=110 and 155+/-10 A respectively) show that their average conformations are rather extended. The length of the statistical segment (twice the persistence length) is b=30 A, which is larger than the usual value (18 A-20 A) for unstructured polypeptide chains. These characteristics are presumably related to the presence of polyproline helices within the polypeptidic backbones. For both proteins, the radius of gyration of the chain cross-section is Rc=2.7+/-0.2A. The glycosylated protein II-1 has similar conformations but the presence of large polyoside sidegroups yields the structure of a branched macromolecule with the same hydrophobic backbone and hydrophilic branches. It is proposed that the unusually extended conformations of these proteins in solution facilitate the capture of plant tannins in the oral cavity.

Finding new posttranslational modifications in salivary proline-rich proteins.

Proline-rich proteins (PRPs) are the most complex family of salivary peptides with distinct isoforms and PTMs. Up to date, only the serine phosphorylation at positions 8, 17, and 22 have been experimentally observed on acidic PRP (aPRPs), and at position 8 on basic PRP1 and 2. The presence of a glucoronyl group at Ser17 was also noticed on aPRP. The main goal of this study was to identify new PTMs and distinct isoforms of salivary PRPs using LC-MALDI-TOF/TOF. Through the salivary peptidome characterization of 20 different subjects from Control, Diabetic, and Head and Neck Cancer groups, it was possible to identify the following species: (i) N-glycosylation sites: two in basic proline-rich protein 2 (bPRP2), one in bPRP3 and one in bPRP4; (ii) O-glycosylation sites: two in bPRP2 and one in aPRP; (iii) other terminal monosaccharide sites: six in bPRP1, two in bPRP2 and two in bPRP3; (iv) other modifications such as N-terminal pyro-Glu (two in bPRP1, six in bPRP2, eight in bPRP3 and nine in bPRP4); (v) phosphorylation in serine, three in bPRP1, one in bPRP2, one in bPRP3 and one in aPRP1; (vi) bPRP1 (allele S, allele M and variant CP5) and bPRP4 (allele M). In summary, salivary peptidome data analysis allowed the identification of 45 new PRP-modified residues, mainly due to glycosylation, phosphorylation and conversion of Gln to pyro-Glu. Moreover, comparing all subject groups, it was noticed a predominance of N-acetyl hexosamine modification on bPRPs in the Head and Neck Cancer patients.

Synthesis and conformational analysis of salivary proline-rich peptide P-B.

The 57-amino acid human salivary polypeptide P-B has been synthesized by the solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc) strategy. The circular dichroism (CD) spectroscopy, Fourier-transform infrared spectroscopy (FTIR) and molecular modeling methods have been used for conformational studies of P-B. Examination of the CD spectra of P-B showed the content of the secondary structure to be independent of temperature over the range 0-60 °C at pH = 7 as well as over the pH range of 2-12 at 37 °C. P-B adopts predominantly unordered structure with locally appearing ß-turns. The cumulative results obtained using the CD and FTIR spectroscopic techniques indicate the percentage of the polyproline type-II (PPII) helix being as low as about 10%. Similarly, the molecular dynamics (MD) simulations reveal only a short PPII helix in the C-terminal fragment of the peptide (Pro(51)-Pro(54)), which constitutes 7%.

Synergistic effect of mixture of two proline-rich-protein salivary families (aPRP and bPRP) on the interaction with wine flavanols.

In this study, we have evaluated by HPLC-DAD, DLS and MALDI-TOF a synergic effect of the coexistence of two salivary-PRP fractions (basic-PRPs and acidic PRPs) on the interaction with flavanols. Results obtained showed noticeable enhancement of the interaction between (epi)catechin and PRPs when both types of proteins are blended. Up to 30 soluble aggregates have been tentatively identified with molecular weight from 4680 to 35,851. (epi)Catechins seem to bind preferentially bPRPs than aPRPs, although the medium size aggregates flavanol-bPRPs formed could favour the interaction with aPRPs giving rise to soluble mixed aggregates.

Interaction between Ellagitannins and Salivary Proline-Rich Proteins.

The first contact of tannins with the human body occurs in the mouth, where some of these tannins are known to interact with salivary proteins, in particular with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for astringency development, but could also affect the biological activities of the tannins. This study gathers information on the relative affinity of the interaction, complex stoichiometry, and tannin molecular epitopes of binding for the interactions between the families of PRPs (bPRPs, gPRPs, and aPRPs) and three representative ellagitannins (castalagin, vescalagin, and punicalagin). These interactions were studied by saturation-tranfer difference NMR and microcalorimetry. The effect of the PRP-ellagitannin interaction on their antioxidant ability was also assessed by ferric reduction antioxidant power (FRAP) assays. The results support a significant interaction between the studied tannins and PRPs with binding affinities in the micromolar range. Punicalagin was always the ellagitannin with higher affinity. aPRPs were the salivary PRPs with higher affinity. Moreover, it was observed that when ellagitannins are present in low concentrations (5-50 µM), as occurs in food, the antioxidant ability of these tannins when complexed with salivary PRPs could be significantly impaired.

Study of human salivary proline-rich proteins interaction with food tannins.

In this work, saturation transfer difference-NMR, isothermal microcalorimetry and molecular dynamics simulations have been used to study the individual interactions between basic, glycosylated and acidic proline-rich proteins (bPRPS, gPRPs, aPRPs) and P-B peptide with some representative food tannins [procyanidin B2, procyanidin B2 3'-O-gallate (B2g) and procyanidin trimer (catechin-4-8-catechin-4-8-catechin)]. Results showed that P-B peptide was in general the salivary protein (SP) with higher affinity whereas aPRPs showed lower affinity to the studied procyanidins. Moreover, B2g was the procyanidin with higher affinity for all SP. Hydrophobic and hydrogen bonds were present in all interactions but the major driving force depended on the procyanidin-SP pair. Furthermore, proline clusters or residues in their vicinity were identified as the probable sites of proteins for interaction with procyanidins. For bPRP and aPRP a significant change to less extended conformations was observed, while P-B peptide did not display any structural rearrangement upon procyanidins binding.

The PBII gene of the human salivary proline-rich protein P-B produces another protein, Q504X8, with an opiorphin homolog, QRGPR.

OBJECTIVES: The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level. DESIGN: To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography; (2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS; and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE. RESULTS: The peptide sequences (I37PPPYSCTPNMNNCSR52, C53HHHHKRHHYPCNYCFCYPK72, R59HHYPCNYCFCYPK72 and H60HYPCNYCFCYPK72) present in the variant protein of P-B were identified. The peptide sequence (G6PYPPGPLAPPQPFGPGFVPPPPPPPYGPGR36) in P-B (or the variant) and sequence (I37PPPPPAPYGPGIFPPPPPQP57) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva. CONCLUSIONS: The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3'-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity.

First evidences of interaction between pyranoanthocyanins and salivary proline-rich proteins.

The contribution of other classes of polyphenol compounds besides tannins to the overall perception of astringency is still poorly understood. So, this work aimed to study the interaction between a family of salivary proline-rich proteins (aPRPs) and representative pyranoanthocyanins in red wines [pyranomalvidin-3-glucoside (vitisin B), pyranomalvidin-3-glucoside-catechol, and pyranomalvidin-3-glucoside-epicatechin] using saturation transfer difference-NMR and MALDI-TOF. For vitisin B KD was of 1.74mM; for pyranomalvidin-3-glucoside-catechol was 1.17mM and for pyranomalvidin-3-glucoside-epicatechin it was 0.87mM. The presence of the flavanol structural unit in the pyranoanthocyanins led to an increase in their interaction with aPRPs. Further, it is also interesting that the values obtained were in the range of KD obtained previously reported for the interaction between the human saliva proline-rich peptides (IB714 and IB937) and procyanidins. Overall, the results obtained suggest that, along with tannins, other polyphenols present in red wine, namely pyranoanthocyanins, could actively contribute to red wine global astringency.

Salivary proline-rich proteins and gluten: Do structural similarities suggest a role in celiac disease?

PURPOSE: Gluten proteins, the culprits in celiac disease (CD), show striking similarities in primary structure with human salivary proline-rich proteins (PRPs). Both are enriched in proline and glutamine residues that often occur consecutively in their sequences. We investigated potential differences in the spectrum of salivary PRPs in health and CD. EXPERIMENTAL DESIGN: Stimulated salivary secretions were collected from CD patients, patients with refractory CD, patients with gastrointestinal complaints but no CD, and healthy controls. PRP isoforms/peptides were characterized by anionic and SDS-PAGE, PCR, and LC-ESI-MS. RESULTS: The gene frequencies of the acidic PRP isoforms PIF, Db, Pa, PRP1, and PRP2 did not differ between groups. At the protein level, PRPs peptides showed minor group differences, but these could not differentiate the CD and/or refractory CDs groups from the controls. CONCLUSIONS AND CLINICAL RELEVANCE: This extensive study established that salivary PRPs, despite similarity to gluten proteins, show no apparent correlation with CD and thus will not serve as diagnostic markers for the disease. The structural basis for the tolerance to the gluten-like PRP proteins in CD is worthy of further exploration and may lead to the development of gluten-like analogs lacking immunogenicity that could be used therapeutically.

endoProteoFASP: a novel FASP approach to profile salivary peptidome and disclose salivary proteases.

The salivary peptidome, which can represent up to 20% of total secreted proteins in human saliva, is highly influenced by proteolytic events. However, the development of strategies to understand the dynamics underlying the generation of salivary peptides has been a challenging task. In order to disclose in more detail the proteolytic events taking place in saliva, we aimed to characterize salivary peptidome and predict salivary proteases by applying, for the first time, a filter-aided sample preparation (FASP) approach to saliva. Thus, as a proof-of-concept of this application, harvested saliva samples from healthy individuals were incubated in 30 kDa cut-off spin filters for 18 or 115 h, at 37 °C, to promote saliva autolysis and the attained peptidome was characterized and compared with the naturally occurring one. In ex vivo conditions, proline-rich proteins, P-B peptide, histatin 1 and statherin were found to be the most susceptible salivary proteins to proteolysis. Peptide fragments were mainly attributed to the activity of cathepsin L1 and K at 18 h, whereas at 115 h, the attained peptide fragments were attributed to the activity of cathepsins K and L1, and MEP1A. Overall, the described endoProteoFASP approach makes the most of saliva׳s own protease pool and avoids the use of synthetic peptides and exogenous proteases to understand the proteolytic events occurring in the oral fluid. Hence, it could be very helpful in future studies targeting the characterization of salivary proteases and peptidome from different pathophysiological conditions.

In vivo interactions between procyanidins and human saliva proteins: effect of repeated exposures to procyanidins solution.

The general accepted mechanism for astringency arises from the interaction between tannins and salivary proteins (SP) resulting in (in)soluble aggregates. By HPLC analysis, it was observed that repeated sips of procyanidins (PC) solution practically depleted aPRPs (∼14%) and statherin (∼2%), and significantly reduced the amount of gPRPs. On the other hand, bPRPs were not significantly affected. In the analysis performed after the last exposure to PC solution, it was seen a significant recovering of the chromatographic peaks corresponding especially to aPRPs (∼74%) and statherin (∼80%). In vitro interaction between SP and PC results in the decrease of the chromatographic peaks of aPRPs and statherin, suggesting that these proteins were involved in the formation of a significant quantity of insoluble complexes. In general, the results suggest that the different families of SP can be involved in different stages of the development of astringency sensation.

Effectiveness of cysteine proteases on protein/pigment film removal.

OBJECTIVE: Theaflavin (TF) from the black tea can react to human salivary proline-rich proteins (PRPs) to form stains on exposed dental surfaces. Here, we employed a model of protein/pigment film using TF and dephosphorylated bovine ß-casein (Dß-CN), which has an extended conformation, similar to that of salivary PRPs, on a sensor surface to assess the efficacy of cysteine proteases (CPs) including papain, stem bromelain, and ficin, on removing TF bound to Dß-CN and the control TF readsorption on the residual substrate surfaces was also measured. METHODS: The protein/pigment complex film was built by using a quartz crystal microbalance with dissipation (QCM-D). The efficacies of CPs were assessed by Boltzman equation model. The surface details were detected by grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles. RESULTS: The efficacy order of CPs on hydrolyzing protein/pigment complex film is ficin>papain>bromelain. The results from grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles demonstrated that TF bound on the Dß-CN was effectively removed by the CPs, and the amount of TF readsorption on both the residual film of the Dß-CN/TF and the Dß-CN was markedly decreased after hydrolysis. CONCLUSION: This study indicates the potential application of the CPs for tooth stain removal and suggests that these enzymes are worthy of further investigation for use in oral healthcare.