巯基蛋白酶水解人全唾液的效率和影响因素
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摘要
目的:探讨三种巯基蛋白酶(菠萝蛋白酶、木瓜蛋白酶和无花果蛋白酶)在不同盐浓度、pH值和温度环境中水解人全唾液生物膜的效率。方法:收集非刺激性的人全唾液(whole saliva, WS),采用消散因子石英晶体微天平(QCM-D)在体外原位、实时和动态监测人非刺激性全唾液在Au石英晶体芯片形成生物膜,用菠萝蛋白酶、木瓜蛋白酶和无花果蛋白酶在不同盐浓度、pH值和温度环境中水解唾液生物膜。采用3倍频和归一化方法记录频率改变(△F)和能量消散因子改变(△D)随时间的变化。用分析软件计算膜的质量减小量(△m)和厚度减小量(△h)。通过△m和△h评价蛋白酶水解唾液生物膜的效果。结果:菠萝蛋白酶在盐浓度0.001~0.01 M时,△m和△h无明显变化,高于0.01 M时,△m和△h随着盐浓度的升高而减小。木瓜蛋白酶和无花果蛋白酶分别在盐浓度为0.005 M和0.05 M时,△m和△h最大,水解效率最高,低于或高于该盐浓度,水解效率都降低。随着pH值的升高,菠萝蛋白酶的△m和△h减小,水解效率降低,即pH 5.9>pH 6.9>pH 7.9(P<0.05);无花果蛋白酶的△m和△h增加,水解效率升高,即pH 5.9Objective: The aim of this paper is to investigate the efficiency of hydrolyzing human whole salivary biofilm by three thiol proteases (bromelain, papain and ficin) in different salt concentration, pH and temperatures. Methods: The whole saliva was collected by unstimulated method. The human unstimulated whole salivary biofilm was formed on the gold electrode surface by quartz crystal microbalance with dissipation (QCM-D) in real time and in situ. Bromelain, papain and ficin were used to hydrolyze the human whole salivary biofilm in different salt concentration, pH and temperatures. The normalization method and third overtone frequency were used to record the frequency shift (△F) and energy dissipation shift (△D) with variation of time. Analysis software was used to calculate the changes of the mass (△m) and the thickness (△h) of the biofilm. The efficiency of hydrolyzing human whole salivary biofilm by proteases were calculated by△m and△h. Result: The△m and△h of bromelain had no change with increasing salt concentration from 0.001 to 0.01M, but decreased with increasing salt concentration above 0.01M. Papain and ficin all had a optional salt concentration,which were 0.005M and 0.05M. The△m and△h of papain and ficin were the highest in this concentration and decreased below and above this concentration. The△m and△h of bromelain decreased with rising the pH, which was pH5.9>pH6.9>pH7.9(P<0.05). The△m and△h of ficin increased with rising the pH, which was pH5.9ary biofilm by these three thiol proteases were effected by salt concentration, pH and temperature. The hydrolysis efficiency were difference among these proteases. The hydrolysis efficiency of ficin was the highest in the neutral and basic pH range, while bromelain has the highest efficiency in the acidic pH range.
引文
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4. Schüpbach P, Oppenheim FG, Lendenmann U, et al. Electron-microscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles in vitro [J]. Eur J Oral Sci,2001 Feb,109(1):60-8.
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29. Gaspers PB, Gast AP, Robertson CR. Enzymes on immobilized substrate surfaces: reaction [J]. J. Colloid Interface Sci,1995,172:518-529.
30. Karajanagi SS, Vertegel AA, Kane RS, et al. Structure and function of enzymes adsorbed onto single-walled carbon nanotubes [J]. Langmuir,2004 Dec 21, 20(26):11594-9.
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32. Smith EL, Kimmel JR, Brown DM. Crystalline papain. II. Physical studies: the mercury complex [J]. J Biol Chem,1954 Apr,207(2):533-49.
33. Sluyterman LA, de Graaf MJ. The effect of salts upon the pH dependence of the activity of papain and succinyl-papain [J]. Biochim Biophys Acta,1972 Feb 28, 258(2):554-61.
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35. Yao Y, Berg EA, Costello CE, et al. Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches [J]. J Biol Chem,2003 Feb 14,278(7):5300-8.
36. Bennick A. Salivary proline-rich proteins [J]. Mol Cell Biochem,1982 Jun 11, 45(2):83–99.
37. Schlesinger DH, Hay DI. Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva [J]. J. Biol. Chem. J Biol Chem,1977 Mar 10,252(5):1689-95.
38. Chitpan M, Wang X, Ho C, et al. Monitoring the binding processes of black tea thearubigin to the bovine serum albumin surface using quartz crystal microbalance with dissipation monitoring [J]. J. Agric. Food Chem,2007,55(25): 10110-10116.
39. Lin FY, Chen CS, Chen WY, et al. Microcalorimetric studies of the interaction mechanisms between proteins and Q-sepharose at pH near the isoelectric point (pI) effects of NaCl concentration, pH value, and temperature [J]. J Chromatogr A,2001 Apr 6,912(2):281-9.
40. Chen J, Sun Y. Modeling of the salt effects on hydrophobic adsorption equilibrium of protein [J]. J Chromatogr A,2003 Apr 11,992(1-2):29-40.
41.查锡良主编.生物化学[M],第7版,北京:人民卫生出版社, 2008, 74-75.
42. Devaraj KB, Kumar PR, Prakash V. Purification, Characterization, and Solvent-Induced Thermal Stabilization of Ficin from Ficus carica [J]. J Agric Food Chem,2008 Dec 10,56(23):11417-23.
43. Kilara A, Shahani KM. Preparation and properties of immobilized papain and lipase [J]. Biotechnol Bioeng,1977 Nov,19(11):1703-14.
44.曲和之,黄露,张国华等.无花果蛋白酶与木瓜蛋白酶酶学性质的比较[J].吉林大学学报, 2008, 46(6):1217-1220.
45. Xie J, Aguilar MI, Hearn MT. High-performance liquid chromatography of amino acids, peptides and proteins. CXXXVIII. Adsorption of horse heart cytochrome c onto a tentacle-type cation exchanger [J]. J Chromatogr A, 1995 Feb 3,691(1-2):263-71.
46. Goheen SC, Gibbins BM. Protein losses in ion-exchange and hydrophobic interaction high-performance liquid chromatography [J]. J Chromatogr A, 2000 Aug 18,890(1):73-80.
47. Fang F, Aguilar MI, Hearn MT. Influence of temperature on the retention behaviour of proteins in cation-exchange chromatography [J]. J Chromatogr A, 1996 Apr 5,729(1-2):49-66.
48. Sathish HA, Kumar PR, Prakash V. Mechanism of solvent induced thermal stabilization of papain [J]. Int J Biol Macromol,2007 Oct 1,41(4):383-90. Epub 2007 Jun 2.
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17. Vacca Smith AM, Bowen WH. In situ studies of pellicle formation on hydroxyapatite discs [J]. Arch Oral Biol. 2000 Apr; 45(4):277-91.
18. Schüpbach P, Oppenheim FG, Lendenmann U, et al. Electron-microscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles in vitro [J]. Eur J Oral Sci. 2001 Feb; 109(1):60-8.
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2. Watts A, Addy M. Tooth discolouration and staining: a review of the literature [J]. Br Dent J,2001 Mar 24,190(6):309-16.
3. Lendenmann U, Grogan J, Oppenheim FG, et al. Saliva and dental pellicle--a review [J]. Adv Dent Res,2000 Dec,14:22-8.
4. Schüpbach P, Oppenheim FG, Lendenmann U, et al. Electron-microscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles in vitro [J]. Eur J Oral Sci,2001 Feb,109(1):60-8.
5. Vacca Smith AM, Bowen WH. In situ studies of pellicle formation on hydroxyapatite discs [J]. Arch Oral Biol,2000 Apr,45(4):277-91.
6. Yao Y, Grogan J, Zehnder M, et al. Compositional analysis of human acquired enamel pellicle by mass spectrometry [J]. Arch Oral Biol,2001 Apr,46(4):293-303.
7. H??k F, Kasemo B, Nylander T, et al. Variations in coupled water, viscoelastic properties, and filmthickneSMSLS of a Mefp-1 protein film during adsorption and croSMSLS-linking: A quartz crystal microbalance with diSMSLSipation monitoring, ellipsometry, and surface plasmon resonance study [J]. Anal. Chem, 2001,73(24): 5796–5804.
8. Kim JT, Weber N, Shin GH, et al. The study of beta-lactoglobulin adsorption on polyethersulfone thin film surface using QCM-D and AFM [J]. J Food Sci,2007 May,72(4):E214-21.
9. Muramatsu H, Dicks JM, Tamiya E, et al. Piezoelectric crystal biosensor modified with protein A for determination of immunoglobulins [J]. Anal Chem,1987,59(23): 2760–2763.
10. Kanazawa KK, Gordon JG. Frequency of a Quartz Microbalance in Contact with Liquid [J]. Anal Chem,1985,57(8): 1770-1771.
11. Thornton S, Garnick J. Comparison of ultrasonic to hand instrument in the removal of subginglval plgue [J]. J Periodontol,1982 Jan,53(1):35-7.
12. Horning GM, Cobb CM, Killoy WJ. Effect of an air-powder abrasive system on root surfaces in periodontal surger [J]. J Clin Periodontol,1987 Apr,14(4):213-20.
13. Joiner A. The bleaching of teeth: A review of the literature [J]. Journal of Dentistry, 2006,34:412–9.
14. Rao MB, Tanksale AM, Ghatge MS, et al. Molecular and biotechnological aspects of microbial proteases [J]. Microbiol Mol Biol Rev,1998 Sep,62(3):597-635.
15. Hardesty JO, Casc?o-Pereira L, Kellis JT, et al. Enzymatic proteolysis of a surface-bound alpha-helical polypeptide [J]. Langmuir,2008 Dec 16, 24(24):13944-56.
16. Kim JH, Roy S, Kellis JT, et al. Protease Adsorption and Reaction on an Immobilized Substrate Surface [J]. Langmuir,2002, 18 (16):6312–6318.
17. Navazesh M. Methods for collecting saliva [J]. Ann N Y Acad Sci,1993 Sep 20, 694:72-7.
18. Kruger NJ. The Bradford method for protein quantitation [J]. Methods Mol Biol, 1994, 32: 9-15.
19.陈国洋,姚江武,陶涛.消散因子石英晶体微天平监测人唾液在金表面形成生物膜的过程[J].口腔医学研究, 2009, 25(1):34-37.
20. Bickerstaff GF. Immobilization of Enzymes and Cells [M]. New Jersey Humana Press Inc,1996: 1-11.
21.王娟娟,晁小练,杨祖培.分子自组装技术的研究现状[J].塑料科技, 2004, (4):42-47.
22.翟怡,张金利,李桦等.自组装单层膜的制备与应用[J].化学进展, 2004, 16(4):477-484.
23. Gaspers PB, Robertson CR, Gast AP. Enzymes on Immobilized Substrate Surfaces: Diffusion [J]. Langmuir,1994,10(8): 2699–2704.
24. Roy S, Thomas JM, Holmes EA, et al. Simultaneous Observation of Enzyme Surface Diffusion and Surface Reaction Using Microfluidic Patterning of Substrate Surfaces [J]. Anal Chem,2005 Dec 15,77(24):8146-50.
25. Fears KP, Latour RA. Assessing the Influence of Adsorbed-State Conformation on the Bioactivity of Adsorbed Enzyme Layers [J]. Langmuir,2009 Jun5,25(24):13926-13933.
26. Esker AR, Brode PF, Rubingh DN, et al. Protease Activity on an Immobilized Substrate Modified by Polymers: Subtilisin BPN' [J]. Langmuir,2000, 16(5):2198-2206.
27. Brode PF, Rauch DS. Subtilisin BPN': activity on an immobilized substrate [J]. Langmuir,1992, 8(5):1325-29.
28. Brode PF 3rd, Erwin CR, Rauch DS, et al. Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN' with enhanced surface stability [J]. J Biol Chem,1994 Sep 23,269(38):23538-43.
29. Gaspers PB, Gast AP, Robertson CR. Enzymes on immobilized substrate surfaces: reaction [J]. J. Colloid Interface Sci,1995,172:518-529.
30. Karajanagi SS, Vertegel AA, Kane RS, et al. Structure and function of enzymes adsorbed onto single-walled carbon nanotubes [J]. Langmuir,2004 Dec 21, 20(26):11594-9.
31. Murachi T, Yasui M, Yasuda Y. Purification and Physical Characterization of Stem Bromelain [J]. Biochemistry,1964 Jan,3:48-55.
32. Smith EL, Kimmel JR, Brown DM. Crystalline papain. II. Physical studies: the mercury complex [J]. J Biol Chem,1954 Apr,207(2):533-49.
33. Sluyterman LA, de Graaf MJ. The effect of salts upon the pH dependence of the activity of papain and succinyl-papain [J]. Biochim Biophys Acta,1972 Feb 28, 258(2):554-61.
34.王晓平.无花果蛋白酶的制备[J].技术与市场, 2008, (11): 136.
35. Yao Y, Berg EA, Costello CE, et al. Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches [J]. J Biol Chem,2003 Feb 14,278(7):5300-8.
36. Bennick A. Salivary proline-rich proteins [J]. Mol Cell Biochem,1982 Jun 11, 45(2):83–99.
37. Schlesinger DH, Hay DI. Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva [J]. J. Biol. Chem. J Biol Chem,1977 Mar 10,252(5):1689-95.
38. Chitpan M, Wang X, Ho C, et al. Monitoring the binding processes of black tea thearubigin to the bovine serum albumin surface using quartz crystal microbalance with dissipation monitoring [J]. J. Agric. Food Chem,2007,55(25): 10110-10116.
39. Lin FY, Chen CS, Chen WY, et al. Microcalorimetric studies of the interaction mechanisms between proteins and Q-sepharose at pH near the isoelectric point (pI) effects of NaCl concentration, pH value, and temperature [J]. J Chromatogr A,2001 Apr 6,912(2):281-9.
40. Chen J, Sun Y. Modeling of the salt effects on hydrophobic adsorption equilibrium of protein [J]. J Chromatogr A,2003 Apr 11,992(1-2):29-40.
41.查锡良主编.生物化学[M],第7版,北京:人民卫生出版社, 2008, 74-75.
42. Devaraj KB, Kumar PR, Prakash V. Purification, Characterization, and Solvent-Induced Thermal Stabilization of Ficin from Ficus carica [J]. J Agric Food Chem,2008 Dec 10,56(23):11417-23.
43. Kilara A, Shahani KM. Preparation and properties of immobilized papain and lipase [J]. Biotechnol Bioeng,1977 Nov,19(11):1703-14.
44.曲和之,黄露,张国华等.无花果蛋白酶与木瓜蛋白酶酶学性质的比较[J].吉林大学学报, 2008, 46(6):1217-1220.
45. Xie J, Aguilar MI, Hearn MT. High-performance liquid chromatography of amino acids, peptides and proteins. CXXXVIII. Adsorption of horse heart cytochrome c onto a tentacle-type cation exchanger [J]. J Chromatogr A, 1995 Feb 3,691(1-2):263-71.
46. Goheen SC, Gibbins BM. Protein losses in ion-exchange and hydrophobic interaction high-performance liquid chromatography [J]. J Chromatogr A, 2000 Aug 18,890(1):73-80.
47. Fang F, Aguilar MI, Hearn MT. Influence of temperature on the retention behaviour of proteins in cation-exchange chromatography [J]. J Chromatogr A, 1996 Apr 5,729(1-2):49-66.
48. Sathish HA, Kumar PR, Prakash V. Mechanism of solvent induced thermal stabilization of papain [J]. Int J Biol Macromol,2007 Oct 1,41(4):383-90. Epub 2007 Jun 2.
1. Watts A, Addy M. Tooth discolouration and staining: a reviewof the literature [J]. British Dental Journal 2001;190:309–16.
2. Andrew Joiner. Tooth colour: a review of the literature [J]. J Dent. 2004; 32 Suppl 1:3-12.
3. Ten Bosch JJ, Coops JC. Tooth color and re?ectance as related to light scattering and enamel hardness [J]. Journal of Dental Research 1995; 74:374–80.
4. Nathoo SA. The chemistry and mechanisms of extrinsic and intrinsic discoloration [J]. J Am Dent Assoc. 1997 Apr; 128 Suppl:6S-10S.
5. Stookey GK, Burkhard TA, Schemehorn BR. In vitro Removal of Stain with Dentifrices [J]. J Dent Res. 1982 Nov; 61(11):1236-9.
6. Sheen S, Banfield N, Addy M. The effect of unstimulated and stimulated whole saliva on extrinsic staining in vitro--a developmental method [J]. J Dent. 2002 Sep-Nov; 30(7-8):365-9.
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