Branched Structures of Rice Amylose Examined by Differential Fluorescence Detection of Side-chain Distribution

Abstract

Analytical methods for branched structures of amylose were developed, employing enzymatic debranching followed by fluorescent labeling/high-performance size-exclusion chromatography (HPSEC). The methods allow differential measurement of size distribution of main- and side-chain of branched molecules. Main-chain distribution was obtained by HPSEC of debranched amylose that was labeled before debranching. By labeling after debranching, both main- and side-chains were detected. Side-chain distribution was obtained by subtracting detector response of the main chain from the response of main plus side chains. Main chains showed little difference in the size distribution from that of amylose. As reported previously, debranching of amylose was incomplete and the percentage of debranching was increased from 33 to 67% by prior hydrolysis with β-amylase. Chain-length distribution of side chains that were released from amylose by enzymatic debranching could be divided into long and short chains, and the latter showed similar, but not identical, distribution to that of amylopectin counterpart. Despite of the similarity, chain-length distributions of β-amylase limit dextrin of amylose were distinct from those of amylopectin prepared from the same starch preparation. Molar percentage, estimated as area% of a chromatogram by fluorescence detection, of A and B chains is considerably higher and lower, respectively, in amylose than that in amylopectin. A-chain fraction was 62% in amylose and 57% in amylopectin, and the amount of short B-chains with lengths comparable to B₁-chains in amylopectin was a half (16%) of that in amylopectin. These results imply that unlike amylopectin, short side-chains of amylose are not organized in a cluster fashion.