Ukr.Biochem.J. 2022; Volume 94, Issue 2, Mar-Apr, pp. 76-84

doi: https://doi.org/10.15407/ubj94.02.076

Analysis of chitosan molecular weight profile by electrophoresis in a porosity step gradient polyacrylamide gel

04.07.2022   Uncategorized   No comments

M. D. Lootsik1*, N. O. Manko1, R. O. Bilyy2,
M. M. Lutsyk (Jr.)2, R. S. Stoika1

1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
2Danylo Halytsky National Medical University, Lviv, Ukraine;
*e-mail: lootsikmaxim@gmail.com

Received: 12 April 2022; Accepted: 01 July 2022

Chitosan is biocompatible and biodegradable natural biopolymer widely applied in various fields of biology, medicine, and pharmacy, however, its effects significantly depend on the degree of polymerization (DP) and the degree of deacetylation (DDA) of polymer chains. Evaluation of the chitosan chain diversity by DP requires the use of a highly expensive method of high-performance size exclusion chromatography. The aim of our study was to determine the molecular weight profile of chitosan specimens by the use of electrophoresis in a porosity step gradient polyacrylamide gel and to evaluate the efficacy of this method in monitoring the purification of chitosan fragments and its derivatives. Two types of step gradient porosity gels were used: 1) gels of layers with acrylamide concentration 2.5, 3.5, 5.0, 10.0, 15.0, 20.0 % w/v for native chitosan or its high molecular fragments; 2) gels of layers with acrylamide concentration 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 % w/v for low molecular chitosan fragments. The main amount of molecules from the chitosan pool was localized in the type 1 gel in the region of 550-40 kDa and distributed among three bands, which in different samples differed significantly in percentage. Electrophoresis of chitosan fragments fractionated by gel permeation chromatography provided a clear separation of medium molecular weight fragments (50–400 kDa) in type 1 gel and of low molecular weight fragments (3–40 kDa) in type 2 gel. Thus the method of chitosan electrophoresis in a step-gradient porosity of polyacrylamide gel was developed which permits to characterize the molecular weight profile of chitosan specimens polymer chains and is effective in monitoring the isolation of chitosan fragments by gel penetration chromatography of molecular weights from 3 to 400 kDa.

Keywords: , , , ,

References:

  1. Dai T, Tanaka M, Huang YY, Hamblin MR. Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Rev Anti Infect Ther. 2011;9(7):857-879. PubMed, PubMedCentral, CrossRef
  2. Kulkarni AD, Patel HM, Surana SJ, Vanjari YH, Belgamwar VS, Pardeshi CV. N,N,N-Trimethyl chitosan: An advanced polymer with myriad of opportunities in nanomedicine. Carbohydr Polym. 2017;157:875-902. PubMed, CrossRef
  3. Zhao D, Yu S, Sun B, Gao S, Guo S, Zhao K. Biomedical Applications of Chitosan and Its Derivative Nanoparticles. Polymers (Basel). 2018;10(4):462. PubMed, PubMedCentral, CrossRef
  4. Mnatsakanyan M, Thevarajah JJ, Roi RS, Lauto A, Gaborieau M, Castignolles P. Separation of chitosan by degree of acetylation using simple free solution capillary electrophoresis. Anal Bioanal Chem. 2013;405(21):6873-6877. PubMed, CrossRef
  5. Wu C, Kao CY, Tseng SY, Chen KC, Chen SF. Determination of the degree of deacetylation of chitosan by capillary zone electrophoresis. Carbohydr Polym. 2014;111:236-244. PubMed, CrossRef
  6. Yomota C, Miyazaki T, Okada S. Determination of the viscometric constants for chitosan and the application of universal calibration procedure in its gel permeation chromatography. Colloid Polym Sci. 1993;271(1):76-82. CrossRef
  7. Berth G, Dautzenberg H. The degree of acetylation of chitosans and its effect on the chain conformation in aqueous solution. Carbohydr Polym. 2002;47(1):39-51. CrossRef
  8. Ottøy MH, Vårum KM, Christensen BE, Anthonsen MW, Smidsrød O. Preparative and analytical size-exclusion chromatography of chitosans. Carbohydr Polym. 1996;31(4): 253-261. CrossRef
  9. Bahrke S, Einarsson JM, Gislason J, Haebel S, Letzel MC, Peter-Katalinić J, Peter MG. Sequence analysis of chitooligosaccharides by matrix-assisted laser desorption ionization postsource decay mass spectrometry. Biomacromolecules. 2002;3(4):696-704. PubMed, CrossRef
  10. Nguyen S, Hisiger S, Jolicoeur M, Winnik FM, Buschmann MD. Fractionation and characterization of chitosan by analytical SEC and 1H NMR after semi-preparative SEC. Carbohydr Polym. 2009;75(9):636-645. CrossRef
  11. Blagodatskikh IV, Kulikov SN, Vyshivannaya OV, Bezrodnykh EA, Tikhonov VE. N-Reacetylated oligochitosan: pH dependence of self-assembly properties and antibacterial activity. Biomacromolecules. 2017;18(5):1491-1498. PubMed, CrossRef
  12. Kang Y, Wu X, Ji X, Bo S, Liu Y. Strategy to improve the characterization of chitosan by size exclusion chromatography coupled with multi angle laser light scattering. Carbohydr Polym. 2018;202:99-105. PubMed, CrossRef
  13. Audy P, Asselin A. Gel elctrophoretic analysis of chitosan hydrolytic products. Electrophoresis. 1992; 13(1): 334-337. PubMed, CrossRef
  14. Lootsik MD, Bilyy RA, Lutsyk MM, Stoika RS. Preparation of chitosan with high blood clotting activity and its hemostatic potential assessment. Biotechnologia Acta. 2015;8(6):32-40. CrossRef
  15. Lootsik MD, Bilyy RO, Lutsyk MM, Manko NO, Navytka SA, Kutsiaba VI, Stoika R. S. Honeybee (Apis mellifera) chitosan: purification, heterogeneity and hemocoagulating activity. Biotechnologia Acta. 2016;9(6):39-49. CrossRef
  16. Immunoglobulin Structure and Classes. Regime of access : https://www.thermofisher.com/ua/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/immunoglobulin-structure-classes.html.
  17. Immunoglobulin A. Regime of access : https://en.wikipedia.org/wiki/Immunoglobulin_A
  18. Ban E, Choi OK, Ryu JC, Yoo YS. Capillary electrophoresis of high-molecular chitosan: the natural carbohydrate biopolymer. Electrophoresis. 2001;22(11):2217-2221. PubMed, CrossRef
  19. Beaudoin ME, Gauthier J, Boucher I, Waldron KC. Capillary electrophoresis separation of a mixture of chitin and chitosan oligosaccharides derivatized using a modified fluorophore conjugation procedure. J Sep Sci. 2005;28(12):1390-1398. PubMed, CrossRef
  20. Wang CY, Hsieh YZ. Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence. J Chromatogr A. 2002;979(1-2):431-438. PubMed, CrossRef
Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.