Biochemistry (Moscow)

, Volume 84, Issue 1, pp 33–39 | Cite as

New Sorbent on the Basis of Covalently Immobilized Lysozyme for Removal of Bacterial Lipopolysaccharide (Endotoxin) from Biological Fluids

  • P. A. LevashovEmail author
  • D. A. Matolygina
  • E. D. Ovchinnikova
  • I. Yu. Adamova
  • O. A. Dmitrieva
  • A. V. Nuzhdina
  • N. S. Pokrovsky
  • N. L. Eremeev


It was demonstrated for the first time that immobilized lysozyme can efficiently remove Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (endotoxins) from solutions. Experimentally confirmed sorption capacity for the developed sorbent was at least 400 ng of endotoxin per ml sorbent. The new sorbent is compatible with the whole human blood and can be potentially used in extracorporeal therapy in the treatment of sepsis.


endotoxin sorption sepsis immobilized lysozyme 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Cohen, J. (2002) The immunopathogenesis of sepsis, Nature, 420, 885–891.CrossRefGoogle Scholar
  2. 2.
    Fleischmann, C., Scherag, A., Adhikari, N. K., Hartog, C. S., Tsaganos, T., Schlattmann, P., Angus, D. C., and Reinhart, K. (2016) Assessment of global incidence and mortality of hospital–treated sepsis. Current estimates and limitations, Am. J. Respir. Crit. Care Med., 193, 259–272.CrossRefGoogle Scholar
  3. 3.
    Danner, R. L., Natanson, C., Elin, R. J., Hosseini, J. M., Banks, S., MacVittie, T. J., and Parrillo, J. E. (1990) Pseudomonas aeruginosa compared with Escherichia coli produces less endotoxemia but more cardiovascular dys–function and mortality in a canine model of septic shock, Chest, 98, 1480–1487.CrossRefGoogle Scholar
  4. 4.
    Opal, S. M. (2010) Endotoxins and other sepsis triggers, Contrib. Nephrol., 167, 14–24.CrossRefGoogle Scholar
  5. 5.
    Ongkudon, C. M., Chew, J. H., Liu, B., and Danquah, M. K. (2012) Chromatographic removal of endotoxins: a bio–process engineer’s perspective, ISRN Chromatogr., 2012, 1–9.CrossRefGoogle Scholar
  6. 6.
    Esteban, E., Ferrer, R., Alsina, L., and Artigas, A. (2013) Immunomodulation in sepsis: the role of endotoxin removal by polymyxin B–immobilized cartridge, Mediators Inflamm., 2013, 1–12.CrossRefGoogle Scholar
  7. 7.
    Anspach, F. B., and Hilbeck, O. (1995) Removal of endo–toxins by affinity sorbents, J. Chromatogr. A, 711, 81–92.CrossRefGoogle Scholar
  8. 8.
    Yaroustovsky, M., Abramyan, M., Komardina, E., Nazarova, H., Popov, D., Plyushch, M., Soldatkina, A., and Rogalskaya, E. (2018) Selective LPS adsorption using polymyxin B–immobilized fiber cartridges in sepsis patients following cardiac surgery, Shock, 49, 658–666.CrossRefGoogle Scholar
  9. 9.
    Orwa, J. A., Govaerts, C., Busson, R., Roets, E., Van Schepdael, A., and Hoogmartens, J. (2001) Isolation and structural characterization of polymyxin B components, J. Chromatogr. A, 912, 369–373CrossRefGoogle Scholar
  10. 10.
    Sedov, S. A., Belogurova, N. G., Shipovskov, S., Levashov, A. V., and Levashov, P. A. (2011) Lysis of Escherichia coli cells by lysozyme: discrimination between adsorption and enzyme action, Colloids Surf. B Biointerfaces, 88, 131–133.CrossRefGoogle Scholar
  11. 11.
    Matolygina, D. A., Osipova, H. E., Smirnov, S. A., Belogurova, N. G., Eremeev, N. L., Tishkov, V. I., Levashov, A. V., and Levashov, P. A. (2015) Determination of bacteriolytic activity and measurement of enzyme sorp–tion by cells in the system of living Lactobacillus plantarum, Mosc. Univ. Chem. Bull., 70, 292–297.CrossRefGoogle Scholar
  12. 12.
    Porath, J., and Axen, R. (1976) Immobilization of enzymes to agar, agarose, and Sephadex supports, Methods Enzymol., 44, 19–45.CrossRefGoogle Scholar
  13. 13.
    Schaumberger, S., Ladinig, A., Reisinger, N., Ritzmann, M., and Schatzmayr, G. (2014) Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus ame–bocyte lysate test: an in vitro study, AMB Express, 4, 1–9.CrossRefGoogle Scholar
  14. 14.
    Schmaldienst, S., Goldammer, A., Spitzauer, S., Derfler, K., Horl, W. H., and Knobl, P. (2000) Local anticoagula–tion of the extracorporeal circuit with heparin and subse–quent neutralization with protamine during immunoad–sorption, Am. J. Kidney Dis., 36, 490–497.CrossRefGoogle Scholar
  15. 15.
    Levashov, P. A., Sedov, S. A., Belogurova, N. G., Levashov, A. V., and Shipovskov, S. (2010) Quantitative turbidimetric assay of enzymatic gram–negative bacteria lysis, Anal. Chem., 82, 2161–2163.CrossRefGoogle Scholar
  16. 16.
    Matolygina, D. A., Dushutina, N. S., Ovchinnikova, E. D., Eremeev, N. L., Belogurova, N. G., Atroshenko, D. L., Smirnov, S. A., Savin, S. S., Tishkov, V. I., Levashov, A. V., and Levashov, P. A. (2018) Enzymatic lysis of living micro–bial cells: a universal approach to calculating the rate of cell lysis in turbidimetric measurements, Mosc. Univ. Chem. Bull., 73, 47–52.CrossRefGoogle Scholar
  17. 17.
    Jager, F. C. (1968) Determination of vitamin E requirement in rats by means of spontaneous haemolysis in vitro, Nutr. Dieta Eur. Rev. Nutr. Diet., 10, 215–223.Google Scholar
  18. 18.
    Levashov, P. A., Sutherland, D. S., Besenbacher, F., and Shipovskov, S. (2009) A robust method of determination of high concentrations of peptides and proteins, Anal. Biochem., 395, 111–112.CrossRefGoogle Scholar
  19. 19.
    Levashov, P., Ovchinnikova, E., Afanasieva, M., Frid, D., Azmuko, A., Bespalova, Zh., Adamova, I., Afanaiseva, O., and Pokrovskii, S. (2012) Affinity sorbent based on trypto–phyl–threonyl–tyrosine for binding of the immunoglobulins G: sorption characteristics and aspects of practical applica–tion, Russ. J. Bioorg. Chem., 8, 46–50.CrossRefGoogle Scholar
  20. 20.
    Laemmli, U. K. (1970) Cleavage of structural proteins dur–ing the assembly of the head of bacteriophage T4, Nature, 227, 680–685.CrossRefGoogle Scholar
  21. 21.
    Hurley, J. C., Nowak, P., Ohrmalm, L., Gogos, Ch., Armaganidis, A., and Giamarellos–Bourboulis, E. J. (2015) Endotoxemia as a diagnostic tool for patients with suspect–ed bacteremia caused by Gram–negative organisms: a meta–analysis of 4 decades of studies, J. Clin. Microbiol., 53, 1183–1191.CrossRefGoogle Scholar
  22. 22.
    Marshall, J. C., Walker, P. M., Foster, D. M., Harris, D., Ribeiro, M., Paice, J., Romaschin, A. D., and Derzko, A. N. (2002) Measurement of endotoxin activity in critically ill patients using whole blood neutrophil dependent chemi–luminescence, Crit. Care, 6, 342–348.CrossRefGoogle Scholar
  23. 23.
    Schwarz, H., Gornicec, J., Neuper, Th., Parigiani, M. A., Wallner, M., Duschl, A., and Horejs–Hoec, J. (2017) Biological activity of masked endotoxin, Sci. Rep., 7, 1–11.CrossRefGoogle Scholar
  24. 24.
    Behre, G., Schedel, I., Nentwig, B., Wormann, B., Essink, M., and Hiddemann, W. (1992) Endotoxin concentration in neutropenic patients with suspected gram–negative sep–sis: correlation with clinical outcome and determination of anti–endotoxin core antibodies during therapy with poly–clonal immunoglobulin M–enriched immunoglobulins, Antimicrob. Agents Chemother., 36, 2139–2146.CrossRefGoogle Scholar
  25. 25.
    Zaninetti, C., Biino, G., Noris, P., Melazzini, F., Civaschi, E., and Balduini, C. L. (2015) Personalized reference inter–vals for platelet count reduce the number of subjects with unexplained thrombocytopenia, Haematologica, 100, 338–340.CrossRefGoogle Scholar
  26. 26.
    Kiseleva, E. A., Afanasieva, O. I., Kosheleva, N. A., and Pokrovsky, S. N. (1996) Immunosorbent for IgG apheresis: an in vitro study, Transfus. Sci., 17, 519–525.CrossRefGoogle Scholar
  27. 27.
    Wetter, L. R., and Deutsch, H. F. (1951) Immunological studies on egg white proteins. IV. Immunochemical and physical studies of lysozyme, J. Biol. Chem., 192, 237–242.Google Scholar
  28. 28.
    Blaha, M., Pit’ha, J., Blaha, V., Lanska, M., Maly, J., Filip, S., and Langrova, H. (2010) Extracorporeal immunoglobu–lin elimination for the treatment of severe myasthenia gravis, J. Biomed. Biotechnol., 2010, 419520.CrossRefGoogle Scholar
  29. 29.
    Daniel, M. P., Gaikwad, V., Verghese, M., Abraham, R., and Kapoor, R. (2015) Serum lysozyme (muramidase) lev–els in intra–abdominal abscesses: an experimental study, Indian J. Surg., 77, 117–119.CrossRefGoogle Scholar
  30. 30.
    Falagas, M. E., and Kasiakou, S. K. (2006) Toxicity of polymyxins: a systematic review of the evidence from old and recent studies, Crit. Care, 10, 1–13.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  • P. A. Levashov
    • 1
    • 2
    Email author
  • D. A. Matolygina
    • 1
    • 2
  • E. D. Ovchinnikova
    • 3
  • I. Yu. Adamova
    • 4
  • O. A. Dmitrieva
    • 3
  • A. V. Nuzhdina
    • 2
  • N. S. Pokrovsky
    • 2
    • 5
  • N. L. Eremeev
    • 1
  1. 1.Lomonosov Moscow State UniversityFaculty of ChemistryMoscowRussia
  2. 2.Bauman Moscow State Technical UniversityInterindustry Engineering Center for Composite MaterialsMoscowRussia
  3. 3.National Medical Research Center of Cardiology, Institute of Experimental CardiologyMinistry of Healthcare of the Russian FederationMoscowRussia
  4. 4.POKARD LtdMoscowRussia
  5. 5.Lomonosov Moscow State UniversityFaculty of Fundamental MedicineMoscowRussia

Personalised recommendations