Extraction, isolation and characterisation of phytoglycogen from su-1 maize leaves and grain

Abstract

Phytoglycogen is a highly branched soluble α-glucan found in plants, particularly those with decreased activity of isoamylase-type starch debranching enzyme, such as sugary-1 (su-1) maize. An improved technique has been designed to extract and isolate phytoglycogen from the grain and leaves of su-1 maize with minimal degradation for structural characterisation. The structures of extracted phytoglycogen samples were analysed using size-exclusion chromatography (SEC, also termed GPC) and transmission electron microscopy (TEM) and compared with the structure of pig liver glycogen. The SEC weight molecular size distributions indicate that the extraction procedure with protease is most effective in obtaining pure phytoglycogen from grain, whereas that without protease at cold temperature followed by purification using a sucrose gradient is more effective for leaf material. The extracted and purified phytoglycogen samples from both grain and leaf contain wide distributions of molecular sizes (analysed by SEC and TEM), with the smallest being "individual" β particles, which collectively form larger α particles; the latter are dominant in the phytoglycogen samples examined here. The results show that phytoglycogen is similar to liver glycogen in both the range of molecular size distribution and in the presence of α particles. Abbreviations* AUC*area under the curve*C*ice-cold tricine buffer treatment at 0-4 C*DAP*days after pollination*DBE*debranching enzyme*DMSO/LiBr*dimethyl sulfoxide containing 0.5% (w/w) lithium bromide*SG*sucrose gradient*IUPAC*International Union of Pure and Applied Chemistry*NMR*nuclear magnetic resonance*P*treatment using protease in tricine buffer at 37 C*Rh*hydrodynamic radius*SEC*size-exclusion chromatography*sh-2*shrunken-2 genetic mutation*su- 1*sugary-1 genetic mutation*TEM*transmission electron microscopy*Vh*hydrodynamic volume*W*warm tricine buffer treatment at 37 C. © 2013 Elsevier Ltd. All rights reserved.