METHODS FOR THE ASSAY OF 1,5-ANHYDRO-D-FRUCTOSE AND ALPHA-1,4-GLUCAN LYASE

1,5-Anhydro-D-arabino-hex-2-ulose (1,5-anhydro-D-fructose, 1,5AnFru), produced by alpha-1,4-glucan lyase (EC 4.2.2.13) acting on starch, gly cogen, or related D-glucose oligo-and polysaccharides as substrate, re acts with alkaline 3,5-dinitrosalicylic acid reagent (DNS) at room tem perature (22 degrees C) within 10 min. The absorbance of the reaction mixture at 550 nm at the end of the reaction was proportional to a 1,5 AnFru content in the range of 0.5 to 16 mu mol (80 mu g to 2.6 mg) mL( -1). 1,5AnFru determined by this colorimetric, one test tube one reage nt method, was in good agreement with that found by H-1-NMR spectrosco py 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 degrees C; therefore, they do not interf ere 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 contr ast to the DNS method, they were not specific. Instead of assaying 1,5 AnFru, the activity of alpha-1,4-glucan lyase was analysed enzymatical ly by quantifying glucose or 4-nitrophenol released using maltose and 4-nitrophenyl alpha-maltopentaoside as substrate, respectively. (C) 19 98 Elsevier Science Ltd.