Momordica charantia seeds are known to contain a galactose specific lectin that has been well characterized. Seed extracts also contain glycosidases such as the β-hexosaminidase, α-mannosidase and α-galactosidase. In the present study, lectin was affinity purified from the seed extracts and protein bodies isolated by sucrose density gradient centrifugation. From the protein bodies, lectin was identified and β-hexosaminidase was isolated by lectin affinity chromatography and subsequently separated from other glycosidases by gel filtration. In the native PAGE, the purified β-hexosaminidase migrated as a single band with a molecular weight of ∼235 kDa and by zymogram analysis using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide substrate it was confirmed as β-hexosaminidase. Under reducing conditions in SDS-PAGE, the purified enzyme dissociated into three bands (Mr 33, 20 and 15 kDa). The prominent bands (20 and 15 kDa) showed immunological cross-reactivity with the human Hexosaminidase B antibody in a western blot experiment. In gel digestion of the purified enzyme, followed by proteomic analysis using tandom MS/MS revealed sequence identity as compared to the genomic sequence of the Momordica charantia with a score of 57 (24% sequence coverage). Additionally, by CD analysis the purified β-hexosaminidase showed 39.1% of α-helix. Furthermore, secondary structure variations were observed in presence of substrate, lectin and at different pH values. Protein body membrane prepared from the isolated protein bodies showed a pH dependent interaction with the purified lectin and mixture of glycosidases.
Keywords: Circular dichroism; Lectin Affigel; Mass spectrometry; Protein bodies; Sucrose density gradient centrifugation; Tandem mass spectroscopy.
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