Molecular Weight Distribution of Whole Starch in Rice Endosperm by Gel-permeation Chromatography

Abstract

Starch is a carbohydrate produced in plants by photosyn-thesis and mainly stored in storage organs (seeds, cotyle-dons, and tubers). Starch is a homopolymer of glucose units composed of amylose, a primarily linear molecule connected by α-1,4 glucosidic bonds, and amylopectin, a highly branched molecule linked via α-1,4 and α-1,6 glucosidic bonds. The higher-order structure of starch is still unknown, but there are two models of the molecular structure of amylopectin: the cluster model 1)2) proposed by many researchers and the building block and the backbone model. 3) Nakamura and Kainuma 4) reviewed these models, explored inconsistencies in the building block and the backbone model, and showed that the cluster model is less inconsistent. According to the cluster model, unit chains of amylopec-tin are organized in a cluster structure, in which branching positions are concentrated in an amorphous region. Clusters consist of amorphous and crystalline regions, and the latter are filled with double helices of α-glucan chains and a few branching points. 5) The nonrandom nature of branching positions has been recognized as the origin of an alternating lamellar structure of crystalline and amorphous regions, and results in multiple clusters connected in a tandem fashion. 6) However, the detailed starch structure, such as arrangement of amylose and amylopectin, and size of amylopectin molecules, remains unknown. 7) An analytical method that uses undegraded whole starch can be used to assess the macrostructure and molecular weight (MW) of starch. However, in most starch structural analyses, starch branches are cleaved by various kinds of amylolytic enzymes with well-established specificities, and the resulting small molecules are analyzed. In previous studies, gel-permeation chromatography (GPC) of whole starch using a Sephacryl S1000 column (Pharmacia Biotech, Uppsala, Sweden) yielded two peaks, one each for amylopectin and amylose in order of decreasing MW. 8)9) GPC of whole starch using a Shodex OH pak KB-G guard column and KB-806 and KB-804 analytical columns (Showa Denko K.K., Tokyo, Japan) also yielded two peaks, one each for amylopectin and amylose. 10)11) Yoo and Jane 11) estimated that the weight-average MW (Mw) of rice amylopectin using high-performance size-exclusion chromatography with multiangle laser-light scattering (HPSEC-MALLS) was 27 × 10 8. GPC analyses of whole corn starch using Toyopearl HW75S ╳ 2, HW65S, and HW55S columns revealed two peaks derived from amylopectin and amylose. 12) Takeda et al. 13) found that when undegraded amylopectin purified from different plant materials was separated by GPC (HW40S, HW50S, and HW75S packed in three layers), only Abstract: Starch is comprised of very large α-glucan molecules composed primarily of linear amylose and highly branched amylopectin. Most methods for analyses of starch structure use hydrolytic enzymes to cleave starch. When undegraded, whole starch structures can be analyzed by gel-permeation chromatography (GPC), but this typically yields a single peak each for amylopectin and amylose. The objective of this study was to stably separate amylopectins in whole starch based on their molecular weight using GPC, and to determine the structure of each peak. When alkali-gelatinized whole starch was applied to GPC columns (Toyopearl HW75S2, HW65S, and HW55S), it was separated into three peaks. Iodine staining and chain length distribution analyses of debranched samples showed that peaks were mainly composed of high-molecular weight (MW) amylopectin consisting of many clusters, low-MW amylopectin consisting of a small number of clusters, and amylose. This is an open-access paper distributed under the terms of the Creative Commons Attribution Non-Commercial (by-nc) License (CC-BY-NC4.0: https://creativecommons.org/licenses/by-nc/4.0/).