Methods for the assay of 1,5-anhydro-d-fructose and α-1,4-glucan lyase

1,5-Anhydro-d-arabino-hex-2-ulose (1,5-anhydro-d-fructose, 1,5AnFru), produced by α-1,4-glucan lyase (EC 4.2.2.13) acting on starch, glycogen, or related d-glucose oligo- and polysaccharides as substrate, reacts with alkaline 3,5-dinitrosalicylic acid reagent (DNS) at room temperature (22 °C) within 10 min. The absorbance of the reaction mixture at 550 nm at the end of the reaction was proportional to a 1,5AnFru content in the range of 0.5 to 16 μmol (80 μg to 2.6 mg) mL−1. 1,5AnFru determined by this colorimetric, one test tube one reagent method, was in good agreement with that found by 1H-NMR spectroscopy and HPLC. The DNS method is also specific as other reducing sugars, such as glucose, maltose maltosaccharides, starch and glycogen do not give a colour with DNS at 22 °C; therefore, they do not interfere in the determination. The DNS method is applicable to lyase assay for both cell-free extracts and purified enzyme. Methods for reducing sugar analyses, based on the reduction of ferric and cupric ions, were examined for 1,5AnFru and they proved to be quantitative but in contrast to the DNS method, they were not specific. Instead of assaying 1,5AnFru, the activity of α-1,4-glucan lyase was analysed enzymatically by quantifying glucose or 4-nitrophenol released using maltose and 4-nitrophenyl α-maltopentaoside as substrate, respectively.