Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracers

Mukhametova, L.I.; Zherdev, D.O.; Kuznetsov, A.N.; Yudina, O.N.; Tsvetkov, Y.E.; Eremin, S.A.; Krylov, V.B.; Nifantiev, N.E.

Авторы: Mukhametova Liliya I., Zherdev Dmitry O., Kuznetsov Anton N., Yudina Olga N., Tsvetkov Yury E., Eremin Sergei A., Krylov Vadim B., Nifantiev Nikolay E.
Журнал: Biomolecules
Том: 14
Номер: 2
Год издания: 2024
Издательство: MDPI
Местоположение издательства: Basel, Switzerland
Первая страница: 170
Последняя страница: 170
DOI: 10.3390/biom14020170
Аннотация: Abstract: 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 munohistochemistry, genetic and cellular ngineering 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 lowreproducibility of the assay results. In this work, we propose the use of a chemically synthesizedsubstrate 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.
Добавил в систему: Мухаметова Лилия Инилевна

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Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracersстатья

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