3种条件下木瓜蛋白酶水解唾液膜的动力学和形态学研究

目的:研究木瓜蛋白酶对人全唾液膜水解的动力学。方法:在不同pH值、离子强度和温度条件下,分别通过消散石英晶体微天平(QCM-D)和原子力显微镜(AFM)观察酶水解唾液膜的频率和表面形态变化。结果:在各种条件下唾液膜被木瓜蛋白酶部分水解,变得薄而光滑。QCM-D和AFM测量证实,在木瓜蛋白酶水解唾液膜的过程中,pH值、离子强度和温度具有协同作用。结论:酶动力学显示木瓜蛋白酶水解唾液膜的过程具有pH值、离子强度和温度依赖性和协同作用的特点。     

消散石英晶体微天平对木瓜蛋白酶水解唾液膜的动力学研究

目的：本实验以人全唾作为唾液获得性膜的体外实验模型,研究不同pH值、离子强度和温度条件下木瓜蛋白酶水解的动力学。方法：通过消散石英晶体微天平在各种条件下观察酶水解唾液膜的频率变化。结果：在各种条件下唾液膜被木瓜蛋白酶部分水解。木瓜蛋白酶与唾液蛋白质的相互作用对pH值、离子强度和温度的改变敏感。在选择和应用酶时,该特性应加以考虑。降低pH值,提高离子强度（1～5mmol／L范围内）和温度,木瓜蛋白酶的催化效率增强。结论：酶动力学显示木瓜蛋白酶水解唾液膜的过程具有pH值、离子强度和温度的依赖性特点。在牙膏、漱口水、义齿清洁剂的配方中利用木瓜蛋白酶的这一特点,有助于获得理想的抗菌复合剂。酶的最佳化尚需更进一步理解反应过程中的调节机制和多重的影响因素。     

不同条件下木瓜蛋白酶对唾液／色素复合膜的清除效果

目的：考察3种条件（离子强度、pH值和温度）下木瓜蛋白酶对羟基磷灰石（hydroxyapaptite,HA）表面人全唾液（whole saliva,WS）／茶黄素（theaflavin,TF）复合膜的水解效率。方法：通过消散石英晶体微天平（quartz crystal microbalance with dissipation,QcM—D）在芯片HA的表面构筑ws／TF复合膜,运用玻尔兹曼方程评估不同离子强度、pH值和温度下酶水解ws／TF复合膜的水解参数。结果：木瓜蛋白酶水解复合膜效率的离子强度依次为10mmol／L〉100mmol／L〉70mmol／L〉50mmol／L〉30mmol／L（P〈0．05）;pH值依次为pH7．O〉pH6．5〉pH7．5（P〈0．05）;温度依次为35℃〉25℃〉20oC（P〈0．05）。结论：木瓜蛋白酶水解HA表面wS／TF复合膜的效率具有离子强度、pH值和温度依赖性。     

pH值、温度和离子强度对色素与人类唾液相互作用的影响

目的:通过表面等离子体共振(surface plasma resonance,SPR)技术在分子水平上原位、实时和动态考察pH值、温度和离子强度对茶黄素(theaflavin,TF)、姜黄素(curcumin,Cur)和矢车菊素(cyanidin,Cy)吸附于人全唾液(human whole saliva,WS)表面的影响。方法:利用表面等离子共振技术和吸附动力学原理,在SPR芯片表面自组装形成人全唾液蛋白质生物膜。通过改变实验条件,在不同的pH值、温度和离子强度条件下监测TF、Cur和Cy吸附于WS表面的响应强度。统计采用双因素方差分析和SNK-q检验,检验水平α=0.05。结果:伴随着pH值和温度的升高,三种色素在人类全唾液表面的响应强度降低,吸附量逐渐减少。当反应体系的离子强度位于5.0～12.5 mmol/L范围内时,吸附量与离子强度呈正相关,而当反应体系的离子强度低于5.0 mmol/L或高于12.5 mmol/L时,吸附量与离子强度呈负相关。结论:静电反应是色素和唾液蛋白质分子之间相互作用的主要驱动力。     

Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH

The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.     

Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH.

The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.     

木瓜蛋白酶水解对β-酪蛋白膜的润湿性和构象的影响

目的：评价木瓜蛋白酶水解对牛β-酪蛋白（β-CN）膜的润湿性和构象的影响。方法：将β-CN通过自组装固定于芯片表面,采用消散因子石英晶体微天平（quartz crystal microbalance with dissipation,QCM-D）测量β-CN膜频率（ΔF）变化。β-CN膜水解前后的润湿性和构象变化分别通过接触角测量仪和掠角红外光谱（GA-FTIR spectra）仪测量。结果：水解后β-CN膜接触角变小,伴随酶的浓度从0.5～12.5mg/L增加,β-CN膜由疏水性向亲水性转变。FTIR测量表明β-CN膜水解后的构象发生了改变。除α-螺旋结构外,β-CN膜含有的二级结构（%）均变小,表明水解反应首先发生与β-CN分子的疏水性C端。疏水性反应和氢键结合是水解反应的主要驱动力。结论：木瓜蛋白酶水解β-酪蛋白β-CN膜导致了润湿性和构象的改变,其生物学意义有待于进一步研究。     

Electrochemical synthesis and in situ spectroelectrochemical characterization of poly(3,4-ethylenedioxythiophene) (PEDOT) in room temperature ionic liquids

The electrochemical synthesis and the charging–discharging reactions of poly(3,4-ethylenedioxythiophene) (PEDOT) in two room-temperature ionic liquids, 1-butyl-3-methyl-imidazolium tetrafluoroborate (BMIMBF₄) and 1-butyl-3-methyl-imidazolium hexafluorophosphate (BMIMPF₆) were studied with cyclic voltammetry, in situ attenuated total reflection Fourier transform infrared (ATR–FTIR) spectroscopy and by in situ UV–Vis spectroelectrochemistry. The structures of the films prepared in ionic liquids was compared to PEDOT films prepared in common organic media. The doping induced changes in absorption of the material both in the mid infrared and in the UV–Vis region were studied and interpreted according to spectra reported for PEDOT films prepared from common organic media.     

Degradation of bovine incisor root collagen in an in vitro caries model.

The effects of pH, ionic strength and proteinases on the destruction of bovine incisor root collagen were studied. Experiments were done with powdered and intact root specimens. Completely demineralized root powder was subjected to solutions of varying pH and ionic strength: (a) 0.1 M acetic acid, pH 4.0, (b) 0.1 M acetic acid + 0.15 M KCl, pH 4.0, (c) 0.1 M Hepes, pH 7.0 or to (d) 0.1 M Hepes + 0.15 M KCl, pH 7.0 at 37 degrees C. The surfaces of intact root specimens were exposed to 0.1 M acetic acid, pH 4.0 (which resulted in erosive lesions) or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0 (which produced subsurface lesions) at 37 degrees C. After incubation, the extracts were analysed for soluble collagen and the insoluble matrices were treated with trypsin at 15 degrees C to determine the denatured collagen. To estimate sensitivity to non-specific proteinases, demineralized root powder was also treated with trypsin under physiological conditions of temperature, pH and ionic strength. The denaturation and subsequent solubilization of collagen material from the fibrils could be influenced by variations in pH and ionic strength but these effects were small when compared to proteolytic degradation under physiological conditions. This supports the hypothesis that, in root caries, destruction of exposed matrix collagen depends largely on the presence and activity of proteinases.     

Salivary glucose clearance, dry mouth and pH changes in dental plaque in man

The aim was to study the effect of different salivary secretion rates on glucose clearance in saliva and on pH changes in dental plaque in man. Eighteen dental students, 21–33 yr old, participated. Dry mouth was induced by injecting methylscopolamine-nitrate submucosally in the labial sulcus. When dry mouth was established, two variables were measured at l h intervals, while the salivary flow was recovering: (1) secretion rate of resting and paraffin wax-stimulated whole saliva, and (2) glucose clearance in saliva after ingestion of a glucose tablet. pH changes in dental plaque were studied in 9 of the 18 subjects after a mouth rinse with 10% glucose at two separate occasions, once before and once after an injection of methylscopolamine-nitrate. Higher glucose concentration levels in saliva were found at low than at normal salivary secretion rate. After logarithmic transformation of the salivary glucose concentration values, a biphasic elimination pattern could be seen, with a steeper initial phase followed by a slower one. A critical value for the secretion rate with respect to the salivary glucose clearance time was found to be 0.14 ml/min for resting and 0.62 ml/min for stimulated whole saliva (mean values). The pH changes in dental plaque after the mouth rinse with glucose at extremely low secretion rate were significantly more pronounced than at normal flow rate. Thus, salivary secretion rate affects both the glucose clearance in saliva and the pH changes in dental plaque in man.     

巯基蛋白酶水解蛋白质／色素复合膜

目的：采用茶黄素（theaflavin,TF）和去磷酸化牛β-酪蛋白（dephosphorylated bovineβ-casein,Dβ-CN）在芯片表面形成蛋白质/色素复合膜的模型,评估3种巯基蛋白酶（cysteine proteases,CPs）,木瓜蛋白酶、菠萝蛋白酶和无花果蛋白酶,对Dβ-C/TF复合膜的清除作用。方法：在消散石英晶体微天平（quartz crystal microbalance with dissipation,QCM-D）上建立Dβ-C/TF复合膜,Boltzman方程评估CPs水解复合膜的质量,水解前后膜变化通过掠角傅里叶变换红外光谱（grazing angle fourier transform infrared spectroscopy spectra,GA-FTIR）。结果：3种CPs水解复合膜的功效由大到小依次为无花果蛋白酶、木瓜蛋白酶、菠萝蛋白酶（P〈0.05）。红外光谱表征结果证实,CPs能够有效地清除结合在Dβ-CN表面的TF。结论：本研究表明,CPs具有在牙色渍清除中潜在的应用价值。在开发口腔保健产品中该类酶值得进一步研究。     

Purification of large quantities of human salivary cystatins S, SA and SN: their interactions with the model cysteine protease papain in a non-inhibitory mode

OBJECTIVES: Our aim was to purify large quantities of human salivary cystatins S, SA and SN in order to determine whether these salivary cystatins have a stable interaction with cysteine proteases at a second binding site, other than the protease active site. This property may affect their availability to act as cysteine protease inhibitors within the oral environment. METHODS: Salivary cystatins S, SA and SN were purified from human submandibular sublingual saliva to homo- geneity by column chromatography. Formation of stable complexes between the model cysteine protease papain in the absence of reductant was assessed by SDS-PAGE and probing Western blots with antibody to human salivary cystatin SN. Proteolytic activity of the complex was determined in the gel after electrophoresis. RESULTS AND CONCLUSIONS: Only cystatin SN (14.3 kD) was found to form a stable complex with papain (22 kD) that could be separated by SDS-PAGE producing a Coomassie stained band at (37 kD). After western transfer this same band (37 kD) cross-reacted with antibody to SN. In the presence of E64, an active site inhibitor of cysteine proteases, the same complex was formed, suggesting that SN is able to bind to papain at a site other than the active site. Activity staining of the gel confirmed that this complex (-E64) retained proteolytic activity. Such complex formation between cystatin SN and cysteine proteases in a non-inhibitory mode may reduce its availability to act as an effective cysteine protease inhibitor in the oral environment.     

绿茶多酚导致口腔收敛性感觉的红外和荧光光谱研究

目的：本研究旨在利用红外和荧光光谱,探索绿茶多酚的典型化合物表没食子儿茶素没食子酸酯（EGCG）与全唾液（WS）和腮腺唾液（PS）结合引起口腔收敛性感觉的分子机制。方法：通过获得的荧光光谱,计算Stern-Volmer（KSV）、双分子猝灭（Kq）、静态猝灭（KS）和表观静态猝灭常数（K）。采用傅立叶变换红外光谱仪测量WS和PS修饰的石英晶体表面吸附EGCG前后的红外光谱。结果：KS大于K表明猝灭类型以静态猝灭为主,并形成了EGCG/唾液基态复合物。WS的KS大于PS提示WS的猝灭程度高于PS。红外光谱表明EGCG吸附唾液的驱动力来源于氢键。结论：EGCG可通过其结构中大量的羟基启动口腔收敛性感觉反应。全唾液较之腮腺唾液有更强的中和能力和拮抗EGCG的生物学作用。     

Effect of zeta potential and surface energy on bacterial adhesion to uncoated and saliva-coated human enamel and dentin

Physicochemical surface characteristics of early plaque-forming bacteria and of human tooth surfaces were measured to establish their role in bacterial adhesion to intact dental tissue slabs. In addition, the influence of an experimental salivary pellicle was evaluated. Strains of S. mutans, S. sanguis, S. salivarius, A. viscosus, and A. odontolyticus showed relatively high surface free energies (range, 99-128 mJ.m-2) and carried a negative surface charge, at both physiological (mu = 0.057) and low (mu = 0.020) ionic strengths of the medium. Very large differences in hydrophobicity were detected when the hexadecane adsorption test was used for measurement. Powdered enamel and dentin were also negatively charged at low ionic strength but were slightly positively charged in the physiological buffer. The surface free energy of enamel and dentin increased upon saliva coating, whereas the surface charge was always negative. The adhesion experiments showed: (1) large differences in the binding of various bacteria to the same surface; (2) an up to 20-fold difference in the binding of the same bacterium to different surfaces, although the binding of some strains was relatively independent of the type of surface or presence of a salivary pellicle; (3) a significant decrease in adhesion when the ionic strength of the medium was lowered, due to increased electrostatic repulsion (however, the adhesion of some bacteria was independent of the ionic strength of the medium); (4) different time-dependent adherence kinetics, depending on both the bacteria and nature of the solid surface; and (5) a propensity for plaque streptococci to bind to uncoated dentin.     

Salivary lysozyme and related parameters of a predominantly Chinese, HIV-infected cohort in Hong Kong

OBJECTIVES: To evaluate the salivary lysozyme concentration, flow rate and pH of a predominantly Chinese, HIV-infected group in Hong Kong, and to compare with an equal number of age and gender-matched HIV-free individuals.STUDY DESIGN: A prospective longitudinal study over a 12-month period of 32 predominantly Chinese, male, HIV-infected group in a hospital setting in Hong Kong. Whole saliva collection by expectoration, lysozyme evaluation by 'lysoplate method'; pH and flow rate evaluation using standard methods and correlation with other clinical parameters using regression analysis. RESULTS: The flow rate and the pH of saliva were lower compared with HIV-free, healthy individuals (both P < 0.0001) and salivary lysozyme concentration of the HIV-infected group was 23% higher compared with the HIV-free group (P < 0.001), though there was no significant difference between the lysozyme output (P > 0.05) expressed as microg min-1. On multiple regression analysis, intravenous drug users had a higher salivary lysozyme concentration compared with the homosexual group (P = 0.0015) though other variables investigated were not significantly related to the salivary lysozyme concentrations.CONCLUSIONS: The significant changes in the flow rate, pH value and lysozyme concentration of whole saliva of the HIV-infected individuals as compared with the HIV-free, healthy individuals, may be due to the disease itself or a combination of factors including the medications used in the disease management.     

The effect of pH,temperature and plaque thickness on the hydrolysis of monofluorophosphate in experimental dental plaque

Monofluorophosphate (MFP), an anti-caries agent commonly used in toothpaste, is known to be degraded to fluoride and orthophosphate by bacterial phosphatases in dental plaque. We have examined the effect of pH, temperature, plaque thickness and some ions on this process. Both natural plaque and artificial microcosm plaque incubated with purified MFP at pH 4-10 showed an optimum pH of approximately 8 for hydrolysis. Diffusion and concomitant hydrolysis were examined in an apparatus in which artificial plaque was held between rigid membranes separating two chambers. When MFP diffused through a plaque of 0.51-mm thickness over 4 h it was almost completely hydrolysed at pH 8, but hydrolysis on diffusion decreased as the pH deviated from 8. MFP in toothpaste extract showed a similar pH susceptibility to hydrolysis, according to the inherent pH of the toothpaste. Hydrolysis of MFP in the toothpaste was reduced by no more than 10% when compared with a matched-pH control, suggesting that other toothpaste ingredients had no major influence on hydrolysis. Transport was slower and hydrolysis at pH 6 more complete the thicker the plaque, but hydrolysis was not significantly slower at 23 degrees C than at 37 degrees C. The addition of various potential activating or inhibiting ions at 0.1 and 1.0 mmol/l had small and non-significant effects on hydrolysis. The results suggest that MFP toothpaste should be formulated and used to maximise enzymic hydrolysis of this complex anion, and that plaque pH control is probably the most important factor.     

Effects of pH, ionic strength, and applied voltage on migration of dental monomers in an organic matrix

Objectives

The application of an electric field has been shown to positively influence the bonding of dentin bonding systems (DBS) by improving adhesive impregnation into dentin. However, the mechanism responsible for this phenomenon has not been completely elucidated. The aim of this study was to clarify the effects of pH, matrix ionic strength, and applied voltage on the migration of commonly used DBS monomers in a model matrix (agarose gel).

Methods

Some common monomers examined were bis-GMA (2,2-bis[4-(2-hydroxy-3-methacryloyloxy propoxy) phenyl] propane); HEMA (2-hydroxyethyl methacrylate); 2-MP (bis[2-(methacryloyloxy) ethyl] phosphate); TCDM [di(hydroxyethyl methacrylate) ester of 5-(2,5,-dioxo tetrahydrofurfuryl)-3-methyl-3-cyclohexenyl-1,2-dicarboxylic acid]; and TEGDMA (triethylene glycol dimethacrylate). Agarose gels poured into a horizontal 10-well electrophoretic cell were used to mimic the collagen fibrils of the dentin organic matrix. The role of pH, matrix ionic strength, and voltage on monomer migration was assayed by modifying the experimental conditions.

Results

Results of experiments performed at pH 3.1, 6.3, 8.5, and 12.3; at low, medium, and high ionic strength; and at 50 and 100 V clearly showed that DBA monomer migration toward both the anode and the cathode can be affected by each of these parameters.

Significance

Migration of acrylic monomers toward the anode or cathode can be achieved as desired by selective choice of pH, ionic strength, and applied voltage. Additional studies are needed to evaluate the synergistic effects of DBS monomer blends on migration in an electric field.     

木瓜蛋白酶水解对β-酪蛋白膜的形态和黏弹性的影响

目的：评价木瓜蛋白酶水解对牛β-酪蛋白（β-CN）膜表面形态和黏弹性的影响。方法：将β-CN通过自组装固定于芯片表面,采用消散因子石英晶体微天平（quartz crystal microbalance with dissipation,QCM-D）测量β-CN膜能量消散因子（△D）和频率（△F）和黏弹性变化。β-CN膜水解前后的表面形态变化通过原子力显微镜（atomic force microscopy,AFM）表征。结果：伴随着酶浓度提高,残余的β-CN膜的黏弹性增加（P＜0．05）。AFM图像显示水解后残余的β-CN膜的厚度和粗糙度均降低。结论：木瓜蛋白酶水解β-酪蛋白β-CN膜的反应具有浓度依赖性特征;水解反应使膜变得较薄而光滑,且更加紧凑和较硬。     

Affinity chromatography of human saliva lysozyme and effect of pH and ionic strength on lytic activity

Lysozyme from human saliva was purified in one step by affinity chromatography on chitin. The recovery, however, was always very low, typically 25–40%. When the cationic exchanger BioRex 70® was used, the enzyme was isolated from saliva with only minor loss of activity, and the product appeared to be 60–70% pure. This partially purified enzyme bound completely and reversibly to chitin and was thereby purified to homogeneity with little loss of activity (< 10%). Both saliva lysozyme and the chicken egg white lysozyme were found to exhibit their highest lytic activity at high pH/low ionic strength (pH 9.0, I = 0.03), and both enzymes required higher ionic strength at lower pH for optimal activity. From isoactivity curves (activity as a function of pH and ionic strength) it was found, however, that the ratio of the specific activity of saliva lysozyme to that of chicken lysozyme varied considerably depending on the assay conditions used. We argue strongly, therefore, against the frequent use of chicken lysozyme as a standard of reference in work on human lysozyme.     

木瓜蛋白酶水解β-酪蛋白膜的动力学研究

目的：采用消散因子石英晶体微天平（QCM-D）原位、实时和动态监测木瓜蛋白酶水解β-酪蛋白生物膜的动力学过程,比较不同浓度酶的水解效率。方法：用β-酪蛋白在QCM-D上建立体外蛋白膜模型,采用不同浓度的木瓜蛋白酶水解膜,观察频率和厚度的改变,运用Boltzmann方程对实验数据进行拟合,单因素方差分析和SNK-q检验比较水解参数B、Δh、V50和C。结果：随着酶浓度增大,膜的厚度Δh减薄,ΔF上升增多,水解参数B增大,C减小,V50缩短（P〈0.05）。结论：在一定浓度范围内水解蛋白生物膜时,酶的水解效率随浓度增大而提高。