A procedure developed in this laboratory for the enzymic determination of glycogen was described earlier (1, 2). Krebs and co-workers
(3) reported a similar procedure at about the same time, and have been employing it for the analysis of carbohydrate in tissue extracts (4). The t,echnique employs an amyloglucosidase CAGS)' enzyme to hydrolyze glycogen to glucose, which is then assayed by t,he glucose oxidase method (5). Our preliminary investigations were performed wit,h aqueous solutions of glycogen; this report will discuss the results obtained by direct analysis of liver carbohyratcs.
The procedure for assaying glycogen by acid hydrolysis
(1 'I was improved and employed as a basis for the evalila-Con of enzymic results. Techniques were developed for assay of liver glycogen obtained by KOH-ethanol fractionation (KOH glycogen), and for direct enzymic analysis of liver homogcnnte (DE method). As a necessary adiunrt, to the development of the DE method, a rrliahlr assay for t,issue gluco+c was established.
RE.4GENTS
AC'S Xcrrc/c~f. Diazyme (Ilot No. F-9273, Miles Clrcmie:rl ('0.) cont,ains glucogenic material which can be readily removed by dialysis. A fihcred aqueous solution containing 5 m g Diazyme per ml is dialyzed against, 10 vol 0.1 M phosphate huffcr, pH 6.0, for 20 hr at 4" with a change of buffer at 6 hr. The dialyzed solution is diluted with buffer to a concentration of 1 mg/ml and st'ored frozen until use.