Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracers

Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracers Full article

Journal

Biomolecules
ISSN: 2218-273X

Output data

Year: 2024, Volume: 14, Number: 2, Article number : 170, Pages count : DOI: 10.3390/biom14020170

Authors

Mukhametova Liliya I. ¹ , Zherdev Dmitry O. ^1,2 , Kuznetsov Anton N. ² , Yudina Olga N. ² , Tsvetkov Yury E. ² , Eremin Sergei A. ¹ , Krylov Vadim B. ² , Nifantiev Nikolay E. ²

Affiliations

1	Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninsky Gory 1/3, 119991 Moscow, Russia
2	Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow, Russia Laboratory of Synthetic Glycovaccines, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow, Russia

Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 μM) and rapid (<30 min) determination of human lysozyme. The proposed protocol was applied to assay human lysozyme in tear samples and resulted in good correlation with the reference assay. The use of synthetic fluorescently labeled tracer, in contrast to natural peptidoglycan, in FP analysis allows for the development of a reproducible method for the determination of lysozyme activity.

Mukhametova L.I. , Zherdev D.O. , Kuznetsov A.N. , Yudina O.N. , Tsvetkov Y.E. , Eremin S.A. , Krylov V.B. , Nifantiev N.E.
Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracers
Biomolecules. 2024. V.14. N2. 170 . DOI: 10.3390/biom14020170 WOS Scopus OpenAlex

≡ Web of science:	WOS:001173051000001
≡ Scopus:	2-s2.0-85185953991
≡ OpenAlex:	W4391404361