HPLC Determination of the Sugar Compositions of the Glycans on the Cationic Peanut Peroxidase

Abstract

Cationic peanut peroxidase has three N-linked glycans. Two glycoforms, CP− and CP+, are known to occur. In this study, the glycans of CP− and CP+ were sequentially separated and identified by trypsin digestion, Bio-Gel P-6 filtration, and reverse phase HPLC. Sugar composition analyses of the glycans were carried out by hydrolysis with TFA, labeling the released sugars with ABEE, and reverse phase HPLC of the ABEE-sugar derivatives. Five different sugars (GlcNAc, Gal, Man, Xyl, Fuc) were found in each of the six glycans investigated. Mannose residues accounted for 31 ± 1.8% of CP− and 40 ± 2.2% of CP+ glycans in term of molar content. The galactose content was 57% lower in CP+ glycans as compared to CP−. The Xyl and Fuc contents were also lower in CP+ than in CP− glycans. A hypothesis made has been confirmed one step further, that is the Gal is one of the terminal sugars of the glycans and the removal of the Gal as a terminal sugar residue in CP− leads to the exposure of Man residue, which is then able to bind to Con-A. Keywords: Cationic peanut peroxidases; glycans; HPLC; sugar compositions