EXHAUSTIVE REMOVAL OF N-GLYCANS FROM ASCORBATE OXIDASE - EFFECT ON THE ENZYMATIC-ACTIVITY AND IMMUNOREACTIVITY

Purified ascorbate oxidase from Cucurbita pepo medullosa has been subj ected to enzymatic deglycosylation using peptide N-glycosidase F. Expe rimental conditions were chosen to obtain efficiently deglycosylated a nd active ascorbate oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to th e peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) s odium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the pre sence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native f orm. Moreover, anti-native ascorbate oxidase antibodies scarcely recog nized deglycosylated protein.