Document Type : Research Paper

Authors

1 Department of Molecular and Cell Biology, Faculty of Science, University of Mazandaran, Babolsar, PC:47416-95447, Mazandaran, Iran.

2 Department of Cancer Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Babol, 47138-18983, Mazandaran, Iran.

10.30498/ijb.2023.339309.3364

Abstract

Background: Today, numerous antimicrobial and anticancer properties have been reported for plant lectins due to their
ability to bind to carbohydrates. The Urtica dioica agglutinin (UDA lectin) is a monomeric, small, and low molecular
weight glycoprotein. It has attracted the attention of many researchers for identification, treatment, and other clinical
purposes.
Objectives: The aim of this study is the optimization of the chitin affinity chromatography based on Sepharose 4B (CNBractivated Sepharose 4B) for the rapid purification of UDA lectin from Urtica dioica rhizome.
Materials and Methods: The chitin ligands were dissolved in 40% Trichloroacetic acid and attached to Sepharose
4B according to the Amersham-Biosciences instructions. The attachment of the ligand to the Sepharose 4B beads was
investigated by Fourier transform infrared (FTIR) spectroscopy. An acidic crude extract of nettle rhizome passes from
chromatographic columns in two sizes with dimensions: 24 x 0.51 cm and 8.44 x 0.86 cm. Quantity and quality of purified
lectin were calculated by the Bradford microplate method: SDS-PAGE gel electrophoresis and human erythrocyte cell
(RBC) hemagglutination, respectively.
Results: The analysis of FTIR spectrograms showed that major changes were observed in the fingerprint regions. Besides,
due to the dissolution of Sepharose 4B and chitin in the aqueous phase, this difference was not significant in the Imine
and Nitrile regions. On the other hand, the comparative results of purification chromatograms showed that increasing
the column length causes a smaller half-width and increases the length of the purified peak. Also, it leads to high-quality
purified UDA lectin, with a molecular weight of almost 12.5 kDa in gel electrophoresis. Hemagglutination activity
on trypsinized red blood cells was displayed, and agglutination of purified UDA lectin started at least at 300 μg.mL-1
concentration.
Conclusion: According to our findings, we suggested that dissolving chitin in the polar solvent of Trichloroacetic acid,
using Sepharose 4B as the beads of a matrix, and increasing the column length might lead to a decrease in the half-width
of the peak. These can increase the purity and concentration of purified UDA lectin, and speed up the purification process.
These findings could be used by researchers to accelerate the purification of UDA lectin in other studies, dealing with drug
delivery systems, ELISA techniques, and cell growth.

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