A linked galactose dehydrogenase/galactose mutarotase assay (Megazyme) was used to quantify the release of galactose or arabinose from xyloglucan or XyGOs. The release of galactose or arabinose led to the stoichiometric reduction of NAD+ to NADH, giving an increase in A340 (ε 6230 M−1 cm−1 at pH 7.0),56 (link) which was read continuously using a Cary 300 spectrophotometer.
A second linked assay (Glucose/Mannose/Fructose detection kit, Megazyme) was used to quantify the release of glucose monosaccharides. The protocol provided in the manufacturer’s instructions was modified for use as a continuous assay. The release of a glucose monosaccharide corresponds stoichiometrically with the reduction of a molecule of NADP+ to NADPH, which leads to an increase in A340 (ε 6220 M−1 cm−1),56 (link) observed continuously using a Cary 300 spectrophotometer. Reactions were carried out in the triethylamine buffer (pH 7.6) provided with the assay kit.
A second linked assay (Glucose/Mannose/Fructose detection kit, Megazyme) was used to quantify the release of glucose monosaccharides. The protocol provided in the manufacturer’s instructions was modified for use as a continuous assay. The release of a glucose monosaccharide corresponds stoichiometrically with the reduction of a molecule of NADP+ to NADPH, which leads to an increase in A340 (ε 6220 M−1 cm−1),56 (link) observed continuously using a Cary 300 spectrophotometer. Reactions were carried out in the triethylamine buffer (pH 7.6) provided with the assay kit.