MICRODETERMINATION OF GLYCOGEN WITH ANTHRONE REAGENT

Abstract

With samples of embryonic skeletal muscle, 20-40 µg wet weight, it was possible to measure 0.08 µg of glycogen within 10% in a final volume of 98.4 µl. Commercial anthrone was washed, dissolved in absolute alcohol, filtered and recrystallized. Glycogen was also repurified. For the working reagent, 20 mg of anthrone was dissolved in 10 ml of 77% (v/v) sulfuric acid. A sample was dispersed in 10 µl water in a small test tube, 90 µl anthrone reagent was added, promptly mixed and the tube plugged with a polyethylene stopper. Triplicate 10 µl portions of water and of dilutions of glycogen were treated the same as the sample. All tubes were heated at 100°C for 15 minutes then cooled and optical density read at 630 mµ in a spectrophotometer. The extinction coefficient for a 1% glycogen solution was 490 ± 3% with 1 cm cell length. Recovery experiments were good. Proteins and pentoses did not interfere significantly. Methods for distinguishing between mono- and polysaccharides are discussed.