The Application Of Galactose Binding Champe Ak Lectin In The Isolation And Detection Of Human Serum Glycoproteins

Author

ANIL AZURA JALALUDIN

Date

2003

Keyword

Artocarpus integer, 'champedak', galactose binding lectin, human serum glycoproteins

Abstract

Galactose-binding lectin from the seeds of champedak (Artocarpus integer) has been an indispensable tool applied to analyse the expression of human serum glycoproteins. Characterisation of the lectin was previously done by studies on its binding specificity and by sequencing the N-terminal. Presently, the CGB lectin was isolated from dried seeds of the fruit, followed by its purification by affinity chromatography. It was then coupled to CNBr-Sepharose matrix to prepare CGB lectin affinity column. Human serum was applied to the CGB lectin column to generate CGB lectin bound fractions. These glycoprotein fractions were heavily dialysed against dH20 and freeze dried to optimum concentrations. Aliquots of the protein fractions were separated further by isoelectric points (pH4-7) and molecular sizes in two dimensional electrophoresis (2-DE) analysis. Valid glycoprotein spots on the 2-DE proteome maps were visualised by staining of the gel slabs with silver, detected by scanning the gel images and quantified by Molecular Analyst computer software. The amount of protein in each of the selected spot was measured as a percentage of volume contribution from all valid spots in a particular gel. Three CGB lectin-reactive proteins detected in all of the gels include hemopexin, IgA α chain, and α2 - HS glycoprotein. Less frequently expressed proteins were α1-ß-glycoprotein and α1-antichymotrypsin. An unknown protein, Unknown B was remarkably observed in most of the cancer samples but no proper identification could be determined without further experiments. No significant difference in expression was observed between the glycoproteins of normal samples with those of cancer patients' pooled sera, but these were mainly because of saturated affinity columns.