A SPECTROPHOTOMETRIC ASSAY FOR ALPHA-MANNOSIDASE ACTIVITY

A simple and versatile spectrophotometric assay for alpha-mannosidase activity, which can be used with unlabelled natural substrate or synth etic substrates, was developed, The reducing mannose released from the substrate by the enzyme is quantitated using glucose oxidase, peroxid ase and o-dianisidine, Using recombinant alpha 1,2-mannosidase obtaine d from Saccharomyces cerevisiae and Man(9) GlcNAc, the spectrophotomet ric assay yielded values of 0.3 mM for K-m and 15 mU/mu g for V-max, w hich are comparable to those obtained using the traditional radiochemi cal assay, The assay was also used to evaluate some alternative oligos accharides as substrates for the enzyme, Man(5)-O(CH2)(8)-COOCH3 shows potential as an alternative synthetic substrate as the enzyme retaine d its specificity for a single alpha 1,2-mannose residue, Kinetic resu lts suggest that the lower 1,3 linked arm of Man(9)GlcNAc is more crit ically involved in substrate recognition than the upper 1,6 linked arm .