The Use of Gas-Liquid Chromatography in the Analysis of Smith Degradation Products from Oligosaccharides

Short Communication

The Journal of Biochemistry, Vol. 63, No. 4, 1968

The Use of Gas-liquid Chromatography in the Analysis of Smith Degradation Products from Oligosaccharides

By HARUKI YAMAGUGHI, TOKUJI IKENAKA and YOSHIO MATSUSHIMA

(From Department of Chemistry, Osaka University College of Science, Toyonaka, Osaka)

(Received for publication, October 17, 1967)

Smith degradation procedure (1) has been were, therefore, converted to their oximes extensively applied to the elucidation of the by heating with hydroxylamine hydrochloride structure of oligosaccharides and glycopeptides and then trimethylsilylated. Standard curves (2, 3). This method includes periodate oxi were obtained for glycerol, erythritol, glycol- dation, reduction of the oxidized product and aldehyde and glyceraldehyde using trimethyl preferential hydrolysis of the sensitive acetal olethane (CH3-C(CH2OH)3) as an internal bonds produced. Hexose residues having free standard (Fig. 1). The height of the peak of hydroxyl groups at C2 and Ca will give ery each hydroxy- and oxo-compound was found thritol and glycolaldehyde when subjected to to be proportional to the concentration. the Smith degradation, hexose residues with The usefulness of this method was shown free hydroxyl groups at Ca and C4 will provide in methyl (ƒÀ-cellobioside from which one mole glycerol and glyceraldehyde, and nonreducing terminals with free hydroxyl groups at C.., Ca, C4 and C6 will give glycerol, glycolaldehyde and formic acid. However, hexose residues without any vicinal hydroxyl groups will not be affected by periodate oxidation and remain as such after hydrolysis. The quantitative analysis of these cleav age products, erythritol, glycerol, glyceralde hyde and glycolaldehyde, therefore, would provide much information about the nature of glycosidic linkages in oligosaccharides. The chromotropic acid procedure after chromato graphic separation has been the usual way of determining glycerol and erythritol (4). In the present study, the method of gas- liquid chromatography was examined for the determination of the Smith degradation pro- FIG. 1. Standard curves of Smith degrada ducts and it was found that this method had tion products. advantage in rapidity and sensitivity. Hy The ratios of the heights of the peaks to the droxy- and oxo-compounds were all trimethyl height of the standard were plotted against the silylated as usual (5). Glycolaldehyde could weight ratios of the samples to the standard. not be quantitatively determined by direct -•ü- Glyceraldehyde, -•œ- Glycolaldehyde, trimethylsilylation. These oxo-compounds -ƒ¢- Glycerol, -•£- Erythritol. 553 553 1 13 Glycolaldehyde Glycolaldehydeand glyceraldehyde Short Communication

each of glycerol and erythritol and two moles TABLE I

of glycolaldehyde were produced by the Smith Results from methyl (ƒÀ-cellobioside by degradation as shown in Fig. 2. the proposed procedure. Methyl (-cellobioside (1.00 mg, 2.81 ƒÊmoles) was oxidized with 1.0 ml of 0.1 M sodium

periodate in the dark at 3°-5°C for 96 hr. The resulting oxidation mixture was reduced with sodium borohydride (60 mg) at 3°-5°C for 24 hr and the excess of the borohydride

was decomposed with acetic acid. The resulting solution was desalted by passing through a column of Amberlite IR-4B (OH form, 6•~ 2 cm) and Dowex 50X8 (H form, 3 x 2 cm). The eluate was collected and subjected to mild hydrolysis with 0.1 N hydrochloric acid over- night at 35°-40°C. After adjusting the total volume of the solution to 10 ml, an aliquot

(3.0 ml) was treated with hydroxylamine hy drochloride (6 mg) at 80°C for 30 min in a sealed tube. The reaction mixture was dried up in vacuo, trimethylsilylated in pyridine (0.5 FIG. 2. Smith degradation products of methyl ml, containing 0.5 mg of trimethylolethane as ƒÀ-cellobioside . an internal standard), and then analyzed by

gas chromatography. The chromatogram is shown in Fig. 3. The results of these analyses are given in Table I and were found to be in good agreement with the calculated values. The amount of trimethylsilylated compounds used for gas chromatography corresponded to 0.3-0.5 , ƒÊg each of the hydroxy- and oxo compounds.

The authors wish to thank Sankyo Co., Ltd. for their kind supply of Taka-diastase.

REFERENCES (1) M. Abdel-Akher, J.K. Hamilton, and F. Smith, J. Am. Chem. Soc., 73, 4691 (1951) (2) R. Montgomery, Y.-C. Wu, and Y.-C. Lee, Biochemistry, 4, 578 (1965) (3) M. Makino and I. Yamashina, J. Biochem., 60, FIG. 3. Gas chromatogram of Smith degrada 262 (1966) tion products of methyl ƒÀ-cellobioside. (4) M. Lambert and A.C. Neish, Can. J. Res., 28B, Column : 15% EGS polyester 0.3 X 200 cm, initial 83 (1950) temperature 110°C. (5) C.C. Sweeley, R. Bentley, M. Makita, and Standard : Trimethylolethane. W.W. Wells, J. Am. Chem. Soc., 85, 2497 (1963)

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