Abstract
Objectives
The ester linkages contained within dental resin monomers (such as Bisphenol A-glycidylmethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA)) are susceptible to hydrolytic degradation by salivary esterases, however very little is known about the specific esterase activities implicated in this process. The objective of this work was to isolate and identify the dominant proteins from saliva that are associated with the esterase activities shown to be involved in the degradation of BisGMA.
Methods
Human whole saliva was collected and processed prior to separation in a HiPrep 16/60 Sephacryl S-200 HR column. The fraction with the highest esterase activity was further separated by an anion exchange column (Mono-Q (10/100G)). Isolated fractions were then separated by gel electrophoresis, and compared to a common bench marker esterase, cholesterol esterase (CE), and commercial albumin which has been reported to express esterase activity. Proteins suspected of containing esterase activity were analyzed by Mass Spectroscopy (MS). Commercially available proteins, similar to the salivary esterase proteins identified by MS, were used to replicate the enzymatic complexes and confirm their degradation activity with respect to BisGMA.
Results
MS data suggested that the enzyme fraction with the highest esterase activity was contained among a group of proteins consisting of albumin, Zn-α2-glycoprotein, α-amylase, TALDO1 protein, transferrin, lipocalin2, and prolactin-induced protein. Studies concluded that the main esterase bands on the gels in each fraction did not overlap with CE activity, and that albumin activity emerged as a lead candidate with significant esterase activity relative to BisGMA degradation, particularly when it formed a complex with Zn-α2-glycoprotein, under slightly basic conditions.
Significance
These enzyme complexes can be used as a physiologically relevant formulation to test the biostability of composite resins.
1
Introduction
An increasing demand for esthetic restorations, continued public concern over the release of mercury from amalgam fillings, the improved mechanical properties of composite resin materials, as well as more and improved adhesive resin systems have fueled the use of composite resins in dentistry .
The combination of BisGMA and the diluent monomer TEGDMA remain two of the most common composite resin monomers still in use today due to their many advantages with respect to rapid hardening within the oral environment and ease of handling and manipulation. Both monomers contain ester linkages linking Bis-phenol-A and triethylene glycol segments to polymerizable vinyl segments. However, it is now well recognized that these ester groups are highly susceptible to hydrolysis by salivary enzymes, and can produce toxic and/or pro-biotic products such as methacrylic acid (MA), triethyleneglycol (TEG) and bishydroxypropoxyphenylpropane (BisHPPP) . Concern over these products and their influence on the function of host cells (for example cell morbidity, cellular adhesive function and inhibition of intracellular biomolecular synthesis) and microorganisms has been raised over the past few decades, however, there has been very little work directed towards adapting the materials toward addressing these challenges. Rather the focus of material development has remained primarily on the mechanical function of the materials . One study has shown that the residue from a bis-phenol diglycidyl ether used in dental composites has resulted in allergic reactions, and much controversy has arisen around the issue of the estrogenic cell response to bisphenol A derivatives . The biodegradation of composite resins has also been reported to lead to a softening of the surface layers of resins and predispose the material to mechanical wear during mastication. Once the softened surface layers are removed, newly exposed material would be prone to further degradation and this can finally lead to the failure of interfacial structures at the margins of the restoration .
Human whole saliva is a complex mixture with salivary proteins and food residues. Salivary proteins have many functions such as digestive, protective, calcium and mineral homeostasis, antibacterial and antifungal function, protease inhibition, lubrication . There are only a very small number of esterases reported to be found in saliva and these are typically involved in food hydrolysis and more recent years are suspected to be involved in the degradation of composite resins. Studies by the Santerre group have demonstrated that human saliva exhibits cholesterol esterase-like (CE) and pseudocholinesterase (PCE) hydrolase activities, which are able to degrade composite resin components . However, there is still very little information published on the actual salivary enzyme proteins related to the CE-like components which are involved in the in vivo process of composite degradation.
In order to mimic salivary esterases and to apply them in assessing new products being developed, leading to enhanced function with respect to the physical stability, ultimate chemical stability of composite resins, and biological character of the degradation products from composite resin interactions with cells and bacteria, the current study has sought to isolate and analyze the predominant components in saliva and to identify the specific source of esterase activity that could be involved in catalyzing the breakdown of polymer resins. In the current study, the commercial monomer BisGMA is used as a model substrate solely for the purpose of identifying sources of esterase activity in saliva relevant to the hydrolysis of esters contained within the resin monomer components of composites. Since the susceptible linkages to chemical hydrolysis in methacrylate based polymers are equivalent to that of the monomeric form in its formulations, using the monomer in solution allows for an accelerated and easy investigation of multiple salivary enzyme species, in addition to minimizing the number of parameters that must be controlled for in the study. For example, it removes considerations of surface area, filler loading, and degree of conversion and cross-linking, all of which can affect the rate of degradation and could hinder the focus of the current study.
2
Materials and methods
2.1
Preparation of human saliva
Healthy human whole saliva (20 mLs) was collected in centrifuge tube from 10 donors, 2 h after breakfast and prior to lunch . All human subjects provided informed consent (protocol #8469, reviewed by the human ethics committee at the University of Toronto). The saliva from each donor was thoroughly homogenized with a Polytron PT-2100 tissue homogenizer (Kinematica, Switzerland) for 1 min and centrifuged at 3700 rpm for 1.0 h at 4 °C (Allegra 6R Centrifuge, Beckman Instruments, Mississauga, ON). The supernatant was filtered through 0.8/0.2 μm syringe filters (Acrodisc, NQ105018/1, Pall Corporation, Cornwall, UK) to remove undissolved particulate. The filtrate from each donor was pooled together; quickly frozen by liquid nitrogen and then lyophilized. All dry saliva samples were stored at −78 °C until required.
2.2
Esterase-like activity (CE) assay
The CE-like esterase activity from the human salivary fractions was measured at 401 nm on a Beckman Coulter DU800 spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA, St. Louis, MO, USA) using 4 mM of para-nitrophenylbutyrate (p-NPB) (Sigma–Aldrich, N-9876) as the substrate . One unit of esterase activity was defined as generating 1 nmol/min of p-nitrophenol from p-nitrophenylbutyrate.
2.3
Isolation of proteins from saliva
Saliva samples were reconstituted to a volume ratio of 10–1 with 40 mM sodium phosphate buffer, pH 7.2. Reconstituted saliva (1.5 mL) was centrifuged at 14,000 rpm for 15 min. The supernatant was injected into a High Performance Liquid Chromatograph system (Waters™ 600 Controller, 996 Photodiode Array Detector) with a HiPrep 16/60 Sephacryl S-200 HR gel filtration column (Amersham Biosciences, 17-1166-01, Baie d’Urfé, PQ, Canada). Gel filtration was performed with 40 mM sodium phosphate buffer, pH 7.2, and a flow rate of 0.5 mL/min. Product recovery was monitored at a wavelength of 230 nm. Fractions (3 mL) were collected and measured for esterase activity, and those fractions with the highest activity were pooled for a given peak area, concentrated by lyophilizing, and then dialyzed in a 3500 MWCO, Slide-A-Lyzer ® Dialysis Cassette (PIERCE, prod# 66110) to remove sodium phosphate using a dialysis buffer made up of 20 mM Tris–HCl, 2 mM CaCl 2 , pH 7.0. The dialyzed fractions were further purified by ion exchange chromatography with a Mono-Q (10/100G) column (Amersham Biosciences, 17-5167-01, Baie d’Urfé, PQ, Canada). Buffer A (20 mM Tris–HCl, 2 mM CaCl 2 , pH 7.0) and Buffer B (0.5 M NaCl, 20 mM Tris–HCl, 2 mM CaCl 2 , pH 7.0) were used to perform a linear gradient elution, with a flow rate of 1.0 mL/min. Dialyzed fractions (1.0–5.0 mL) were injected into the HPLC system (Waters™ 600 Controller, 996 Photodiode Array Detector) using carrier Buffer A for the initial 10 min. From 10 to 12 min, Buffer B was introduced in a linear gradient, increasing up to 25%, followed by its continued introduction from 25 to 100% over 12–50 min. The system was kept at 100% Buffer B for a 10 min elution time, and then was equilibrated with Buffer A for 30 min prior to the next injection. Fractions were collected with a Pharmacia ® Fraction Collector using a 1 min fraction interval, and the esterase activity was measured for each fraction. The fractions with elevated esterase activity levels were kept in −78 °C until required for further analysis.
2.4
Gel electrophoresis and mass spectroscopy
Fractions containing esterase activity from the gel filtration column preparation were concentrated using an Amicon Ultra-15 5,000MWC centrifugal filter device (Millipore Corporation catalog #UFC900524) and mixed with native PAGE sample loading buffer containing 0.03% (w/v) bromophenol blue, 0.5 M Tris/HCl (pH 6.8), 30% glycerol, and then directly loaded onto two pieces of a 12% polyacrylamide gel. Gels were run at 200 V in a Mini-PROTEAN 3 Electrophoresis system for 1 h. Esterase activity was identified by soaking the gel in 50 mM pH 6.0 sodium phosphate staining solution with 0.04% Fast Blue RR salt (Sigma Cat. No. F-0500) and 0.02% ß-naphthylbutyrate (Sigma Cat. No. N8125) for 1 h with gentle agitation in the dark. Gels were fixed in 12% (w/v) trichloroacetic acid (Sigma Cat No. T8657) and destained in 10% acetic acid . A second gel was stained by Coomassie blue to stain for non-specific protein.
The fractions containing esterase activity from the Mono-Q column separation were also run on 12% native PAGE and 12% SDS-PAGE in order to isolate proteins prior to mass spectrometry (MS), define protein molecular weight and obtain information on the denatured protein state. Stained bands were excised and sent for mass spectroscopy (MS) analysis at the Advanced Protein Technology Centre at the Hospital for Sick Children (Toronto, ON, Canada) and the Proteomics Core Facility for Molecular and Cellular Biology Research, Sunnybrook & Women’s College Health Sciences Centre (Toronto, ON, Canada). The protein bands were cut into small pieces and dehydrated in acetonitrile, then dried in a vacuum centrifuge. The protein was reduced by incubating with 50 μL of 10 mM dithiotreitol (DTT) in 100 mM NH 4 HCO 3 for 30 min at 56 °C. Samples were allowed to cool to room temperature and excess DTT solution was removed. 50 μL of 20 mM iodoacetamide in 100 mM NH 4 HCO 3 was added into the protein samples and incubated for 30 min at room temperature in the dark to alkylate the sulfhydryl group of the cysteine residue. The excess liquid was removed and the gel pieces were washed with 200 μL of 100 mM NH 4 HCO 3 and completely dried in a vacuum centrifuge. The gel pieces were swollen in a digestion buffer containing 40 μL of 50 mM NH 4 HCO 3 and 12.5 ng/μL of trypsin (Promega, sequencing grade) in an ice bath for 30 min. The supernatant was removed and replaced with 20 μL 50 mM NH 4 HCO 3 . The samples were digested at 37 °C overnight. Peptides were extracted from the gel pieces by shaking with 50 μL of 5% formic acid twice, then 50 μL of 5% formic acid in 30% acetonitrile once (20 min for each extraction) at room temperature. The volume of the peptide extract solution was reduced to about 20 μL by vacuum centrifuge. The peptide solution was injected into an LC–MS/MS system (Agilent 1100 HPLC-chip and 6340 ion trap MSD system, Agilent Technologies). Raw MS/MS data files were matched against an NCBInr human subset database, using the Spectrum Mill MS Proteomics Workbench (Agilent Technologies). Proteins with two or more peptides identified from the MS/MS search were reported on.
2.5
Removal of albumin from salivary fractions of interest
In order to investigate the nature of esterase activity associated with albumin, salivary samples were processed to remove albumin and then the samples, with and without albumin, were characterized for composition and esterase character. The Fractions of interest were rapidly thawed and maintained on ice. The buffer was changed to a ProteoExtract™ binding buffer (Calbiochem Cat. No. 122641 ProteoExtract ® Albumin Removal Kit, Maxi) using Ultra-4 10 K Centrifugal filter devices (Millipore Corporation catalog #UFC801024). Samples were centrifuged in a Beckman GPR centrifuge at 3000 rpm and 4 °C, washed with 4 mL ProteoExtract™ binding buffer, and then concentrated to approximately 0.75 mL. The concentrated sample was loaded onto a ProteoExtract™ column (Calbiochem Cat. No. 122641 ProteoExtract ® Albumin Removal Kit, Maxi) and the flow-through fraction was collected while the columns were eluted twice with 1 mL of binding buffer. All flow-through fractions were pooled together, concentrated with Amicon Ultra-15 5,000MWC centrifugal filter devices, and reconstituted with 20 mM Tris–HCl pH 7.2, 2 mM CaCl 2 . This latter purification work was performed by staff at Lelial Proteiomics Inc. Toronto, ON.
2.6
Esterase activity from commercial human salivary α-amylase preparations
Amylase was identified as an enzyme whose presence corresponded with gel fractions containing high esterase activity. Hence, it was desired to determine whether there was any potential esterase function specifically associated with this enzyme. The esterase activity of amylase (Sigma A-1031) was measured with p-NPB. Amylase activity was assayed by the dinitrosalicylic acid method . 1% (w/v) starch in 50 mM pH 7.0 sodium phosphate buffer was added into amylase samples and incubated at 37 °C for 5 min. A 3,5-dinitrosalicylic acid (DNSA) solution containing 1.0% DNSA, 0.4 M NaOH, 30% sodium potassium tartrate was added into equal volumes of the incubation mixture and boiled for 10 min at 100 °C to stop the enzymatic reaction. The mixture was cooled down by tap water, and a 2.0 ml aliquot was taken to measure the absorbance at 540 nm. One unit of amylase activity was defined as the amount of enzyme releasing 1 μmol glucose per minute at 37 °C pH 7.0. Commercial glucose solutions (0.1–2.0 mM) were used to produce a standard curve. Either the specific α-amylase inhibitor Triticum aestivum (Sigma A-3535) or esterase inhibitor PMSF (phenylmethyl sulfonyl fluoride, 2 mM) (Sigma P-7626) were added to amylase samples and incubated at 37 °C for 30 min before measuring the amylase or esterase activity, respectively.
Purification of the amylase from proteins containing esterase activity was carried out by dissolving approximately 109 mg of α-amylase (Sigma A-1031) from human saliva in 10 mL Milli-Q filtered water, desalted and concentrated to about 2.5 mL by Ultra-15 centrifugal filter (MW cut: 10,000). 250 μL samples were applied into a FPLC (Pharmacia) system with Mono-Q HR (10/10) (Pharmacia), anion-exchange column, eluted for 10 min at a flow rate of 1.0 mL/min. The mobile phase consisted of Buffer A (0.02 M Tris/HCl-0.002 M CaCl 2 , pH 7.2), followed by a linear gradient of 0 to 100% of Buffer B (0.02 M Tris/HCl-0.002 M CaCl 2 , 0.5 M NaCl, pH 7.2) over 40 min. Subsequently, the column was then eluted with 100% Buffer B for 10 min, followed by re-equilibration with Buffer A for 30 min between runs. The eluted solution was monitored at a wavelength of 230 nm and fractions were collected in 1 min intervals. Where chromatographic peaks were observed, fractions were pooled, and esterase activity with respect to p-NPB was measured. Two active fractions of interest were processed by the Advanced Protein Technology Center at the Hospital for Sick Children and analyzed by MS.
2.7
Identification of albumin complexes with esterase activity on native gel
In order to investigate the contribution of albumin protein complexes to esterase activity in saliva, 20 μg of the commercially available single proteins: Zn-α2-glycoprotein (Znα2G), α-amylase, transferin, or lactotransferrin were individually mixed with 20 μg albumin (CalBiochem Cat. 126654) in 20 mM, Tris–HCl pH 7.2 buffer. All mixtures and single protein controls were incubated at 4 °C for overnight, speed vacuum dried, re-dissolved in sample loading buffer, loaded onto a 4–12% native gel, and run in a cold room for 45 min. All gels were then stained with 1% 2-naphthyl-butyrate for an hour under dark conditions. Single protein controls for, Znα2G, lactotransferrin, lipocalin, or albumin were assayed under the same conditions.
2.8
Biodegradation of BisGMA
For the time course study, BisGMA monomer (Esschem, Linwood, PA, USA) was prepared in methanol and diluted with 0.02 M Tris/HCl-0.002 M CaCl 2 , 0.005 M MgCl 2 , pH 7.2 to a desired concentration. This preparation was then added to enzyme solution (see below) containing 100 μg of albumin and yielding a final BisGMA concentration 0.5 × 10 −4 M and total reaction volume 25 μL. Samples were incubated at 37 °C for 24, 48, and 72 h. 25 μL of acetonitrile was added to denature the enzymes and deactivate its esterase activity at the end of the incubation period. All samples were kept in −70 °C until HPLC analysis.
Incubation enzyme solution containing either 100 μg albumin, 20 μg Znα2G, or a combined mixture of 100 μg albumin with 20 μg Znα2G were individually prepared in 0.02 M Tris/HCl-0.002 M CaCl 2 , 0.005 M MgCl 2 , pH 7.2 buffer and allowed to stand at 4 °C overnight prior to use. BisGMA was added into each protein solution to make up the final BisGMA concentration (0.5 × 10 −4 M) and yield a total reaction volume 25 μL. At the end of the 48 h degradation period, 25 μLs of acetonitrile were added to denature the enzymes and cease activity. All samples were stored at −70 °C until required for analysis.
To study the effect of pH on esterase activity, incubation enzyme solutions containing 100 μg albumin were individually prepared in 0.02 M Tris/HCl, 0.002 M CaCl 2 , 0.005 M MgCl 2 , pH buffers adjusted to 6.3, 7.2, 8.0 or 8.8 using HCl and NaOH. BisGMA monomer was prepared in methanol and added into each protein solution to make up the final BisGMA concentration 0.5 × 10 −4 M ([MeOH] < 2 vol%) and a reaction volume 25 μL (note: the presence of MeOH at this concentration did not affect esterase activity). Samples were incubated at 37 °C for 72 h and subsequently, 25 μLs of acetonitrile were added to denature the protein solutions and cease esterase activity. All samples were stored at −70 °C until required for analysis.
Comparison between Cholesterol esterases (CE, Toyobo, COE-313) activity and optimized albumin esterase activity were also investigated in 0.02 M Tris/HCl-0.002 M CaCl 2 , 0.005 M MgCl 2 , pH 8.8 buffer solution at different concentration of the proteins. The enzymatic activity of the CE incubation solutions was adjusted to 10 units/mL with respect to p-NPB substrates. Albumin incubation solutions contained 2, 4, or 10 mg/mL of albumin. The control contained the buffer solution without proteins. Prior to incubating BisGMA with enzyme solutions, stability studies were conducted to assess the effect of the monomer on the enzymatic activity; solution volumes were kept identical to that of the biodegradation studies. Based on the CE stability experiment, the CE containing groups were replenished daily (24 h) in order to maintain the esterase activity near 10 units/mL. Buffer solution was added in equivalent volume to the control and albumin samples to maintain a consistent volume among all three conditions. All solutions were sterile filtered using a 0.22 μm filter (Millipore SLGP033RS). 50 μL of a BisGMA methanol solution (with BisGMA concentration of 2.5 mM) was added into each protein solution to make up the final BisGMA concentration (0.5 × 10 −4 M) and yield a total reaction volume of 2.5 mL. The final methanol concentration in the incubated solutions was less than 2 vol%. The experiment was run with triplicate sample groups to allow for statistical analysis. At specific time points (0, 1, 2, 3 and 4 days), 500 μL samples of the incubation solution were removed and an equal volume of methanol was added to denature the esterase protein and stop the hydrolysis. All samples were stored at −70 °C until required for analysis.
2.9
HPLC analysis of degradation products
Incubation solutions from the BisGMA biodegradation studies ( n = 3) were analyzed using high performance liquid chromatography (HPLC). Each sample was centrifuged at 14,000 rpm for 30 min, and the supernatant was injected into a Luna C18 (2) (phenomenex ® 00G-4252-E0) column. The sample was eluted at a flow rate 1.0 mL/min for 20 min, using a linear gradient beginning from (70:30) methanol (solution A): pH 6.8, 20 mM ammonium acetate (solution B) to (100:0), respectively. This was followed by a 100% methanol elution for 5 min. The column was equilibrated in a 70:30 ratio of solution A: solution B for 30 min before the next injection. The retention time for BisHPPP (terminal degradation product from the hydrolysis of BisGMA) was 5.43 min. The peak fractions were collected and analyzed by mass spectroscopy at the Molecular and Cellular Biology Research Sunnybrook & Women’s College Health Sciences Center. The amount of BisHPPP was calculated from a standard curve equation, r 2 = 0.9991, covering a mass range from 1.8 to 171.5 ng.
2.10
Statistical analysis
Statistical analysis was performed using the SPSS program (version 20.0). One-way analysis of variance (ANOVA) and Turkey’s multiple comparison tests were performed to determine the effect of incubation time and condition on the amount of BisHPPP released. Data are all plotted with standard deviation of the mean ( N = 3). Independent sample t -test was used to determine the statistical significance of any differences between the mean values of two groups. For such analysis, Levene’s test for homogeneity of the variances was conducted. The significance threshold for all analyses was set to α = 0.05.
2
Materials and methods
2.1
Preparation of human saliva
Healthy human whole saliva (20 mLs) was collected in centrifuge tube from 10 donors, 2 h after breakfast and prior to lunch . All human subjects provided informed consent (protocol #8469, reviewed by the human ethics committee at the University of Toronto). The saliva from each donor was thoroughly homogenized with a Polytron PT-2100 tissue homogenizer (Kinematica, Switzerland) for 1 min and centrifuged at 3700 rpm for 1.0 h at 4 °C (Allegra 6R Centrifuge, Beckman Instruments, Mississauga, ON). The supernatant was filtered through 0.8/0.2 μm syringe filters (Acrodisc, NQ105018/1, Pall Corporation, Cornwall, UK) to remove undissolved particulate. The filtrate from each donor was pooled together; quickly frozen by liquid nitrogen and then lyophilized. All dry saliva samples were stored at −78 °C until required.
2.2
Esterase-like activity (CE) assay
The CE-like esterase activity from the human salivary fractions was measured at 401 nm on a Beckman Coulter DU800 spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA, St. Louis, MO, USA) using 4 mM of para-nitrophenylbutyrate (p-NPB) (Sigma–Aldrich, N-9876) as the substrate . One unit of esterase activity was defined as generating 1 nmol/min of p-nitrophenol from p-nitrophenylbutyrate.
2.3
Isolation of proteins from saliva
Saliva samples were reconstituted to a volume ratio of 10–1 with 40 mM sodium phosphate buffer, pH 7.2. Reconstituted saliva (1.5 mL) was centrifuged at 14,000 rpm for 15 min. The supernatant was injected into a High Performance Liquid Chromatograph system (Waters™ 600 Controller, 996 Photodiode Array Detector) with a HiPrep 16/60 Sephacryl S-200 HR gel filtration column (Amersham Biosciences, 17-1166-01, Baie d’Urfé, PQ, Canada). Gel filtration was performed with 40 mM sodium phosphate buffer, pH 7.2, and a flow rate of 0.5 mL/min. Product recovery was monitored at a wavelength of 230 nm. Fractions (3 mL) were collected and measured for esterase activity, and those fractions with the highest activity were pooled for a given peak area, concentrated by lyophilizing, and then dialyzed in a 3500 MWCO, Slide-A-Lyzer ® Dialysis Cassette (PIERCE, prod# 66110) to remove sodium phosphate using a dialysis buffer made up of 20 mM Tris–HCl, 2 mM CaCl 2 , pH 7.0. The dialyzed fractions were further purified by ion exchange chromatography with a Mono-Q (10/100G) column (Amersham Biosciences, 17-5167-01, Baie d’Urfé, PQ, Canada). Buffer A (20 mM Tris–HCl, 2 mM CaCl 2 , pH 7.0) and Buffer B (0.5 M NaCl, 20 mM Tris–HCl, 2 mM CaCl 2 , pH 7.0) were used to perform a linear gradient elution, with a flow rate of 1.0 mL/min. Dialyzed fractions (1.0–5.0 mL) were injected into the HPLC system (Waters™ 600 Controller, 996 Photodiode Array Detector) using carrier Buffer A for the initial 10 min. From 10 to 12 min, Buffer B was introduced in a linear gradient, increasing up to 25%, followed by its continued introduction from 25 to 100% over 12–50 min. The system was kept at 100% Buffer B for a 10 min elution time, and then was equilibrated with Buffer A for 30 min prior to the next injection. Fractions were collected with a Pharmacia ® Fraction Collector using a 1 min fraction interval, and the esterase activity was measured for each fraction. The fractions with elevated esterase activity levels were kept in −78 °C until required for further analysis.
2.4
Gel electrophoresis and mass spectroscopy
Fractions containing esterase activity from the gel filtration column preparation were concentrated using an Amicon Ultra-15 5,000MWC centrifugal filter device (Millipore Corporation catalog #UFC900524) and mixed with native PAGE sample loading buffer containing 0.03% (w/v) bromophenol blue, 0.5 M Tris/HCl (pH 6.8), 30% glycerol, and then directly loaded onto two pieces of a 12% polyacrylamide gel. Gels were run at 200 V in a Mini-PROTEAN 3 Electrophoresis system for 1 h. Esterase activity was identified by soaking the gel in 50 mM pH 6.0 sodium phosphate staining solution with 0.04% Fast Blue RR salt (Sigma Cat. No. F-0500) and 0.02% ß-naphthylbutyrate (Sigma Cat. No. N8125) for 1 h with gentle agitation in the dark. Gels were fixed in 12% (w/v) trichloroacetic acid (Sigma Cat No. T8657) and destained in 10% acetic acid . A second gel was stained by Coomassie blue to stain for non-specific protein.
The fractions containing esterase activity from the Mono-Q column separation were also run on 12% native PAGE and 12% SDS-PAGE in order to isolate proteins prior to mass spectrometry (MS), define protein molecular weight and obtain information on the denatured protein state. Stained bands were excised and sent for mass spectroscopy (MS) analysis at the Advanced Protein Technology Centre at the Hospital for Sick Children (Toronto, ON, Canada) and the Proteomics Core Facility for Molecular and Cellular Biology Research, Sunnybrook & Women’s College Health Sciences Centre (Toronto, ON, Canada). The protein bands were cut into small pieces and dehydrated in acetonitrile, then dried in a vacuum centrifuge. The protein was reduced by incubating with 50 μL of 10 mM dithiotreitol (DTT) in 100 mM NH 4 HCO 3 for 30 min at 56 °C. Samples were allowed to cool to room temperature and excess DTT solution was removed. 50 μL of 20 mM iodoacetamide in 100 mM NH 4 HCO 3 was added into the protein samples and incubated for 30 min at room temperature in the dark to alkylate the sulfhydryl group of the cysteine residue. The excess liquid was removed and the gel pieces were washed with 200 μL of 100 mM NH 4 HCO 3 and completely dried in a vacuum centrifuge. The gel pieces were swollen in a digestion buffer containing 40 μL of 50 mM NH 4 HCO 3 and 12.5 ng/μL of trypsin (Promega, sequencing grade) in an ice bath for 30 min. The supernatant was removed and replaced with 20 μL 50 mM NH 4 HCO 3 . The samples were digested at 37 °C overnight. Peptides were extracted from the gel pieces by shaking with 50 μL of 5% formic acid twice, then 50 μL of 5% formic acid in 30% acetonitrile once (20 min for each extraction) at room temperature. The volume of the peptide extract solution was reduced to about 20 μL by vacuum centrifuge. The peptide solution was injected into an LC–MS/MS system (Agilent 1100 HPLC-chip and 6340 ion trap MSD system, Agilent Technologies). Raw MS/MS data files were matched against an NCBInr human subset database, using the Spectrum Mill MS Proteomics Workbench (Agilent Technologies). Proteins with two or more peptides identified from the MS/MS search were reported on.
2.5
Removal of albumin from salivary fractions of interest
In order to investigate the nature of esterase activity associated with albumin, salivary samples were processed to remove albumin and then the samples, with and without albumin, were characterized for composition and esterase character. The Fractions of interest were rapidly thawed and maintained on ice. The buffer was changed to a ProteoExtract™ binding buffer (Calbiochem Cat. No. 122641 ProteoExtract ® Albumin Removal Kit, Maxi) using Ultra-4 10 K Centrifugal filter devices (Millipore Corporation catalog #UFC801024). Samples were centrifuged in a Beckman GPR centrifuge at 3000 rpm and 4 °C, washed with 4 mL ProteoExtract™ binding buffer, and then concentrated to approximately 0.75 mL. The concentrated sample was loaded onto a ProteoExtract™ column (Calbiochem Cat. No. 122641 ProteoExtract ® Albumin Removal Kit, Maxi) and the flow-through fraction was collected while the columns were eluted twice with 1 mL of binding buffer. All flow-through fractions were pooled together, concentrated with Amicon Ultra-15 5,000MWC centrifugal filter devices, and reconstituted with 20 mM Tris–HCl pH 7.2, 2 mM CaCl 2 . This latter purification work was performed by staff at Lelial Proteiomics Inc. Toronto, ON.
2.6
Esterase activity from commercial human salivary α-amylase preparations
Amylase was identified as an enzyme whose presence corresponded with gel fractions containing high esterase activity. Hence, it was desired to determine whether there was any potential esterase function specifically associated with this enzyme. The esterase activity of amylase (Sigma A-1031) was measured with p-NPB. Amylase activity was assayed by the dinitrosalicylic acid method . 1% (w/v) starch in 50 mM pH 7.0 sodium phosphate buffer was added into amylase samples and incubated at 37 °C for 5 min. A 3,5-dinitrosalicylic acid (DNSA) solution containing 1.0% DNSA, 0.4 M NaOH, 30% sodium potassium tartrate was added into equal volumes of the incubation mixture and boiled for 10 min at 100 °C to stop the enzymatic reaction. The mixture was cooled down by tap water, and a 2.0 ml aliquot was taken to measure the absorbance at 540 nm. One unit of amylase activity was defined as the amount of enzyme releasing 1 μmol glucose per minute at 37 °C pH 7.0. Commercial glucose solutions (0.1–2.0 mM) were used to produce a standard curve. Either the specific α-amylase inhibitor Triticum aestivum (Sigma A-3535) or esterase inhibitor PMSF (phenylmethyl sulfonyl fluoride, 2 mM) (Sigma P-7626) were added to amylase samples and incubated at 37 °C for 30 min before measuring the amylase or esterase activity, respectively.
Purification of the amylase from proteins containing esterase activity was carried out by dissolving approximately 109 mg of α-amylase (Sigma A-1031) from human saliva in 10 mL Milli-Q filtered water, desalted and concentrated to about 2.5 mL by Ultra-15 centrifugal filter (MW cut: 10,000). 250 μL samples were applied into a FPLC (Pharmacia) system with Mono-Q HR (10/10) (Pharmacia), anion-exchange column, eluted for 10 min at a flow rate of 1.0 mL/min. The mobile phase consisted of Buffer A (0.02 M Tris/HCl-0.002 M CaCl 2 , pH 7.2), followed by a linear gradient of 0 to 100% of Buffer B (0.02 M Tris/HCl-0.002 M CaCl 2 , 0.5 M NaCl, pH 7.2) over 40 min. Subsequently, the column was then eluted with 100% Buffer B for 10 min, followed by re-equilibration with Buffer A for 30 min between runs. The eluted solution was monitored at a wavelength of 230 nm and fractions were collected in 1 min intervals. Where chromatographic peaks were observed, fractions were pooled, and esterase activity with respect to p-NPB was measured. Two active fractions of interest were processed by the Advanced Protein Technology Center at the Hospital for Sick Children and analyzed by MS.
2.7
Identification of albumin complexes with esterase activity on native gel
In order to investigate the contribution of albumin protein complexes to esterase activity in saliva, 20 μg of the commercially available single proteins: Zn-α2-glycoprotein (Znα2G), α-amylase, transferin, or lactotransferrin were individually mixed with 20 μg albumin (CalBiochem Cat. 126654) in 20 mM, Tris–HCl pH 7.2 buffer. All mixtures and single protein controls were incubated at 4 °C for overnight, speed vacuum dried, re-dissolved in sample loading buffer, loaded onto a 4–12% native gel, and run in a cold room for 45 min. All gels were then stained with 1% 2-naphthyl-butyrate for an hour under dark conditions. Single protein controls for, Znα2G, lactotransferrin, lipocalin, or albumin were assayed under the same conditions.
2.8
Biodegradation of BisGMA
For the time course study, BisGMA monomer (Esschem, Linwood, PA, USA) was prepared in methanol and diluted with 0.02 M Tris/HCl-0.002 M CaCl 2 , 0.005 M MgCl 2 , pH 7.2 to a desired concentration. This preparation was then added to enzyme solution (see below) containing 100 μg of albumin and yielding a final BisGMA concentration 0.5 × 10 −4 M and total reaction volume 25 μL. Samples were incubated at 37 °C for 24, 48, and 72 h. 25 μL of acetonitrile was added to denature the enzymes and deactivate its esterase activity at the end of the incubation period. All samples were kept in −70 °C until HPLC analysis.
Incubation enzyme solution containing either 100 μg albumin, 20 μg Znα2G, or a combined mixture of 100 μg albumin with 20 μg Znα2G were individually prepared in 0.02 M Tris/HCl-0.002 M CaCl 2 , 0.005 M MgCl 2 , pH 7.2 buffer and allowed to stand at 4 °C overnight prior to use. BisGMA was added into each protein solution to make up the final BisGMA concentration (0.5 × 10 −4 M) and yield a total reaction volume 25 μL. At the end of the 48 h degradation period, 25 μLs of acetonitrile were added to denature the enzymes and cease activity. All samples were stored at −70 °C until required for analysis.
To study the effect of pH on esterase activity, incubation enzyme solutions containing 100 μg albumin were individually prepared in 0.02 M Tris/HCl, 0.002 M CaCl 2 , 0.005 M MgCl 2 , pH buffers adjusted to 6.3, 7.2, 8.0 or 8.8 using HCl and NaOH. BisGMA monomer was prepared in methanol and added into each protein solution to make up the final BisGMA concentration 0.5 × 10 −4 M ([MeOH] < 2 vol%) and a reaction volume 25 μL (note: the presence of MeOH at this concentration did not affect esterase activity). Samples were incubated at 37 °C for 72 h and subsequently, 25 μLs of acetonitrile were added to denature the protein solutions and cease esterase activity. All samples were stored at −70 °C until required for analysis.
Comparison between Cholesterol esterases (CE, Toyobo, COE-313) activity and optimized albumin esterase activity were also investigated in 0.02 M Tris/HCl-0.002 M CaCl 2 , 0.005 M MgCl 2 , pH 8.8 buffer solution at different concentration of the proteins. The enzymatic activity of the CE incubation solutions was adjusted to 10 units/mL with respect to p-NPB substrates. Albumin incubation solutions contained 2, 4, or 10 mg/mL of albumin. The control contained the buffer solution without proteins. Prior to incubating BisGMA with enzyme solutions, stability studies were conducted to assess the effect of the monomer on the enzymatic activity; solution volumes were kept identical to that of the biodegradation studies. Based on the CE stability experiment, the CE containing groups were replenished daily (24 h) in order to maintain the esterase activity near 10 units/mL. Buffer solution was added in equivalent volume to the control and albumin samples to maintain a consistent volume among all three conditions. All solutions were sterile filtered using a 0.22 μm filter (Millipore SLGP033RS). 50 μL of a BisGMA methanol solution (with BisGMA concentration of 2.5 mM) was added into each protein solution to make up the final BisGMA concentration (0.5 × 10 −4 M) and yield a total reaction volume of 2.5 mL. The final methanol concentration in the incubated solutions was less than 2 vol%. The experiment was run with triplicate sample groups to allow for statistical analysis. At specific time points (0, 1, 2, 3 and 4 days), 500 μL samples of the incubation solution were removed and an equal volume of methanol was added to denature the esterase protein and stop the hydrolysis. All samples were stored at −70 °C until required for analysis.
2.9
HPLC analysis of degradation products
Incubation solutions from the BisGMA biodegradation studies ( n = 3) were analyzed using high performance liquid chromatography (HPLC). Each sample was centrifuged at 14,000 rpm for 30 min, and the supernatant was injected into a Luna C18 (2) (phenomenex ® 00G-4252-E0) column. The sample was eluted at a flow rate 1.0 mL/min for 20 min, using a linear gradient beginning from (70:30) methanol (solution A): pH 6.8, 20 mM ammonium acetate (solution B) to (100:0), respectively. This was followed by a 100% methanol elution for 5 min. The column was equilibrated in a 70:30 ratio of solution A: solution B for 30 min before the next injection. The retention time for BisHPPP (terminal degradation product from the hydrolysis of BisGMA) was 5.43 min. The peak fractions were collected and analyzed by mass spectroscopy at the Molecular and Cellular Biology Research Sunnybrook & Women’s College Health Sciences Center. The amount of BisHPPP was calculated from a standard curve equation, r 2 = 0.9991, covering a mass range from 1.8 to 171.5 ng.
2.10
Statistical analysis
Statistical analysis was performed using the SPSS program (version 20.0). One-way analysis of variance (ANOVA) and Turkey’s multiple comparison tests were performed to determine the effect of incubation time and condition on the amount of BisHPPP released. Data are all plotted with standard deviation of the mean ( N = 3). Independent sample t -test was used to determine the statistical significance of any differences between the mean values of two groups. For such analysis, Levene’s test for homogeneity of the variances was conducted. The significance threshold for all analyses was set to α = 0.05.
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