Elaboration of a method of lysozyme purification from Human saliva
- 作者: Krenev I.A.1, Berlov M.N.1
-
隶属关系:
- Institute of Experimental Medicine
- 期: 卷 28, 编号 2 (2025)
- 页面: 40-47
- 栏目: Biological chemistry
- URL: https://ogarev-online.ru/1560-9596/article/view/281070
- DOI: https://doi.org/10.29296/25877313-2025-02-06
- ID: 281070
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详细
Introduction. Growing antibiotic resistance dictates the need for search for alternative antimicrobial drugs. Antimicrobial polypeptides, including lysozyme, are among prospective prototypes. Existing methods of human lysozyme purification are less elaborated in comparison with methods of lysozyme purification from other species sources.
The aim of the study. Development of a simple and rapid method of lysozyme purification from human saliva, which can be performed in laboratories equipped for research of peptides and low-molecular proteins.
Material and methods. Human saliva was treated with NaCl, centrifuged, filtered through filter with pores diameter 0.45 μm, used for two-steps solid-phase extraction on C18 cartridges (elution with 20% and 60% acetonitrile), reversed-phase high-performance liquid chromatography on C18 column in acetonitrile gradient and re-chromatography. Homogeneity and molecular weight were confirmed by SDS-PAGE electrophoresis. Enzyme activity was confirmed in turbidimetric analysis in the presence of Micrococcus lysodeikticus. The presence of antimicrobial activity was confirmed by electrophoresis under acidic conditions with subsequent gel overlay against Listeria monocytogenes.
Results. Saliva treatment with 62.5—1000 mM NaCl did not affect total protein content in the supernatants after centrifugation. Treated with 0.5 M NaCl and filtered saliva was used for solid-phase extraction. After the separation of components eluted with 20% acetonitrile, components eluted with 60% acetonitrile were used for RP-HPLC. As the result, a major peak was obtained and the fractions used for further purification and analysis. The sample contained a protein with the molecular weight of lysozyme. The protein possessed muramidase activity towards Micrococcus lysodeikticus cell walls and antimicrobial activity towards L. monocytogenes.
Conclusions. In the present study, a simple method of lysozyme purification from human saliva was elaborated (about 5 μg of lysozyme from 1 mL of saliva). The protocol preserves essential biological properties of lysozyme.
作者简介
I. Krenev
Institute of Experimental Medicine
编辑信件的主要联系方式.
Email: il.krenevv13@yandex.ru
ORCID iD: 0000-0001-7970-3291
SPIN 代码: 1411-0962
Post-graduate Student, Junior Research Scientist, Department of General Pathology and Pathological Physiology
俄罗斯联邦, Acad. Pavlov Str., 12, Saint Petersburg, 197022M. Berlov
Institute of Experimental Medicine
Email: berlov.mn@iemspb.ru
ORCID iD: 0000-0001-5191-0467
SPIN 代码: 9006-6127
Ph.D. (Biol.), Senior Research Scientist, Department of General Pathology and Pathological Physiology
俄罗斯联邦, Acad. Pavlov Str., 12, Saint Petersburg, 197022
参考
- Ventola C.L. The Antibiotic Resistance Crisis: Part 1: Causes and Threats. P & T. 2015;40(4): 277–283.
- Ventola C.L. The Antibiotic Resistance Crisis: Part 2: Management Strategies and New Agents. P & T. 2015; 40(5): 344–352.
- Ghosh C., Sarkar P., Issa R., Haldar J. Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance. Trends in Microbiology. 2019; 27(4): 323–338. doi: 10.1016/j.tim.2018.12.010.
- Fleming A. On a Remarkable Bacteriolytic Element Found in Tissues and Secretions. Proceedings of the Royal Society of Lon-don. Series B. 1922; 93: 306–317. doi: 10.1098/rspb.1922.0023.
- Ragland S.A., Criss A.K. From Bacterial Killing to Immune Modulation: Recent Insights into the Functions of Lysozyme. PLoS Pathogens. 2017; 13(9): e1006512. doi: 10.1371/jour-nal.ppat.1006512.
- Ferraboschi P., Ciceri S., Grisenti P. Applications of Lysozyme, an Innate Immune Defense Factor, as an Alternative Antibiotic. Antibiotics (Basel). 2021; 10(12): 1534. doi: 10.3390/antibiotics10121534.
- Shahmohammadi A. Lysozyme Separation from Chicken Egg White: a Review. European Food Research and Technology. 2018; 244: 577–593. doi: 10.1007/s00217-017-2993-0.
- Liu B.L., Ooi C.W., Ng I.S. et al. Effective Purification of Lysozyme from Chicken Egg White by Tris(hydroxyme-thyl)amino-methane Affinity Nanofiber Membrane. Food Chemistry. 2020; 327: 127038. doi: 10.1016/j.food-chem.2020.127038.
- Yao X., Du T., Guo J. et al. Extraction and Characterization of Lysozyme from Salted Duck Egg White. Foods. 2022; 11(22): 3567. doi: 10.3390/foods11223567.
- Mohamed R., Campbell J.L., Cooper-White J. et al. The Impact of Saliva Collection and Processing Methods on CRP, IgE, and Myoglobin Immunoassays. Clinical and Translational Medicine. 2012; 1(1): 19. doi: 10.1186/2001-1326-1-19.
- Jenzano J.W., Hogan S.L., Lundblad R.L. Factors Influencing Measurement of Human Salivary Lysozyme in Lysoplate and Turbidimetric Assays. Journal of Clinical Microbiology. 1986; 24(6): 963–967. doi: 10.1128/jcm.24.6.963-967.1986.
- Bradford M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry. 1976; 72: 248–254. doi: 10.1006/abio.1976.9999.
- Schägger H., von Jagow G. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Pro-teins in the Range from 1 to 100 kDa. Analytical Biochemistry. 1987; 166(2): 368–379. doi: 10.1016/0003-2697(87)90587-2.
- Mörsky P. Turbidimetric Determination of Lysozyme with Micrococcus lysodeikticus Cells: Reexamination of Reaction Conditions. Analytical Biochemistry. 1983; 128(1): 77–85. doi: 10.1016/0003-2697(83)90347-0.
- Lehrer R.I., Rosenman M., Harwig S.S. et al. Ultrasensitive Assays for Endogenous Antimicrobial Polypeptides. Journal of Immunological Methods. 1991; 137(2): 167–173.
- doi: 10.1016/0022-1759(91)90021-7.
- Panyim S., Chalkley R. The Heterogeneity of Histones. I. A Quantitative Analysis of Calf Histones in Very Long Polyacrylamide Gels. Biochemistry. 1969; 8(10): 3972–3979. doi: 10.1021/bi00838a013.
- Schipper R.G., Silletti E., Vingerhoeds M.H. Saliva as Research Material: Biochemical, Physicochemical and Practical Aspects. Archives of Oral Biology. 2007; 52(12): 1114–1135. doi: 10.1016/j.archoralbio.2007.06.009.
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