BIOCHEMISTRY
The Structure of the Aerobacter aerogenes A3 (S1 ) Polysaccharide. 11. Sequence Analysis and Hydrolysis Studies" H. E. Conrad, J. R. Barnburg,? J. D. Epley, and T. J. Kindtf
made up of repeating sequences of the tetrasaccharide shown. The acidic tri- and disaccharides are derived from this tetrasaccharide by the hydrolytic removal of one and two D-glucose residues, respectively. Assignments of the anomeric configurations are based on susceptibility of the oligosaccharides to cleavage by specific enzymes and on proton magnetic resonance data. On periodate oxidation the polysaccharide consumes 4 moles of periodate with the formation of 1 mole of formic acid for each anhydrotetrasaccharide unit, as expected from the proposed structure. The first-order rate constant for hydrolysis of the Lfucosyl bond of the polysaccharide in 1 N sulfuric acid at 100" is 300-fold greater than that for the D-glucuronosyl bond and 10- to 20-fold greater than those for the Dglucosyl bonds. The relationship between the complexity of polysaccharide structures and the possible mechanisms for their biosynthesis is discussed.
Quantitative radiochemical methods for determination of reducing equivalents and for radiochromatographic analysis of oligosaccharide mixtures have been developed for use in structural studies of the slime polysaccharide from Aerohucter uerogenes, A 3(S1). Partial acid hydrolysis of the polysaccharide yields a single neutral disaccharide, identified as cellobiose, and three glucuronic acid containing oligosaccharidesa di-, a tri-, and a tetrasaccharideeach of which has L-fucose at the reducing terminal. Sequence analysis, taken with the previous methylation data [Sandford, P. A., and Conrad, H. E. (1966), Biochernistry 5, 15081, shows that the A3(S1) polysaccharide is ABSTRACT :
1/94
-6D-glucose-D-glucuronic
L
1a 3
acid-t,-fucose-
1
l
D-gluzose
1
I
-n
T
he concept that the complex heteropolysaccharides are made up of repeating oligosaccharide sequences is widely accepted (Barker et ul., 1958a,b; How et ul., 1964) but has received experimental documentation in only a few instances (Staub and Tinelli, 1960; Robbins and Uchida, 1962; Osborn et ul., 1964; Jann et ul., 1965; Luderitz and Westphal, 1966). For a large number of plant gums and mucilages (Smith and Montgomery, 1959; Whistler, 1959) and microbial polysaccharides (Stacey and Barker, 1960; How et al., 1964) the available data contradict the repeating unit concept. An earlier report by Aspinall et ul. (1956) indicated that the acidic slime polysaccharide from Aerobacter uerogenes, A3(S1), which is composed of D-glucose, D-glucuronic acid, and Lfucose, is a very complex nonrepeating structure. A reexamination of this polysaccharide using improved methylation methodology (Sandford and Conrad, 1966) showed that the D-glucose, D-glucuronic acid, and L-fucose are present in a simple molar ratio of
2808
IT.
2:1:1, respectively, and that all of the D-glucuronic acid is linked through C-4, all of the L-fucose is linked through C-3, one-half of the D-glucose is at branch points linked through both C-4 and C-6, and the remaining D-glucose is at nonreducing ends of the polysaccharide. This paper describes a sequence analysis of the A3(S1) polysaccharide based on a study of the oligosaccharides obtained on partial hydrolysis. The data show that the polysaccharide has the following simple tetrasaccharide repeating unit. D-glucose '1
2
-%-glucose
J.
I
