Sequence specific analysis of the heterogeneous glycan chain from peanut peroxidase by H-1-NMR spectroscopy

The cationic peanut peroxidase is a complex enzyme consisting of a heme gro up, two calcium ions and three complex carbohydrate chains at positions Asn 60, 144 and 185. Details of the heme and calcium ligation, necessary for ox idation, have recently been revealed from the three-dimensional structure o f the peroxidase. However. the three glycans that may be important for the stability of the enzyme as well as its activity were not resolved. In order to determine the configuration of one of these glycans, PNGase A was used to cleave the glycan from the enzyme at Asn-144. This glycan was studied by two dimensional H-1-NMR spectroscopy to identify the sugar linkages. The r esults indicated a glycan structure comprising a Man alpha 1-6(Xyl beta 1-2 )Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta core but with an add itional Man alpha 1-3 appendage linked to Man3. The glycan also appeared to be heterogeneous as was noted from a single terminating galactose being li nked to approximately 20-25% glycan. (C) 2000 Elsevier Science Ltd. All rig hts reserved.