720 NATURE May 8, 1948 Vol. lbl Separation and Identification of Methylated slippery elm mucilage• and beet araban•, and in on the Paper Chromatogram some instances have found indications of the presence of other sugar derivatives. For example, 2-methyl THE identification of the products of hydrolysis of and 3: 4: 6-trimethylmannose were ob• a methylated composed of many tained from methylated cherry gum, and free different sugar residues is a tedious and difficult task, from methylated slippery elm mucilage. especially when the polysaccharide is available only F. BROWN in small quantities. We have found, however, that E. L. HmsT many of the methylated sugars can be readily sep• L. HOUGH arated and identified on the paper chromatogram, J. K. N. JONES using the same apparatus and solvents as described University, Bristol. H. WADMAN for the separation of the simple sugars1 .s. The University, Edinburgh. chromatogram is allowed to run until the solvent University, Manchester. has advanced about 40 em. from the starting line. March 10. The paper is then dried and sprayed with ammoniacal 1 Partridge, S.M., 158, 270 (1946). silver nitrate, and on warming, the positions of the 'Flood, A. E., Hirst, E. L.,and Jones, J. K. N., Nature, 180,86 (1947). sugars are indicated by brown spots (except in the • Jones, J. K. N., J . Ohem. Soc., 1055 (1947). 'Glll R . E., Hirst, E. L., and Jones, J. K. N., J. Ohem. Soc., 1025 case of tetramethylfructopyranose, which reduces (1946).1 ammoniacal silver nitrate only very slightly). As a • Hirst, E . L., and Jones, J. K. N., forthcoming publication. standard, we use tetramethyl D-, which moves rapidly on the chromatogram. The distance the sugar travels is measured from the starting line Electrophoretic Behaviour of Modified to the centre, not the edge, of the sugar spot, since the size of the spot and its leading edge vary with Ovalbumins the concentration of sugar solution used. The figure IT has been shown recently by Linderstr0m-Lang Ra given in the accompanying table refers to the and Ottesen1 that ovalbumin is transformed, by ratio between the distance the sugar travels and the the action of a bacterial enzyme from Bacillus distance through which the tetramethyl D-glucose subtiliB, into a protein that crystallizes as rectangular has moved. (We use this in preference to the Rp plates from ammonium sulphate. In an earlier paper2, value, which tends to vary with the distance the MacPherson, Moore and Longsworth report that an sugar has advanced from the starting line.) ovalbumin component, A 2 (characterized by its mobility in the electric field), increased at the expense of another one, A 10 during stor• S11bstance RG Substance .Ro age of a salt-free, isoelectric solution under toluene. It therefore seemed of interest to Galactose 0·075 2 : 3-Dimethyl 0·63 Arabinose 0·135 2 : 3 : 4·Trimethyl galactose 0·64 compare the electrophoretic behaviour of 0 ·15 2 : 4-Dimethyl xylose 0·66 the enzyme-modified ovalbumin with the 4-Methyl galactose 0·165 2 : 4 : 6·Trlmethyl galactose 0 ·67 6-Methyl galactose 0·185 2 : 3-Dimethyl xylose 0 ·74 A 1-form of the original protein. , 0 ·19 2 : 4 : 6-Trlmethyl glucose 0·755 Ovalbumin recrystallized three times3 was 2-Methyl galactose 0 ·205 3 : 4 : 6-Trlmethyl 0·795 , 0·21 2 : 3 : 6-Trimethyl glucose 0 ·805 used as starting material. With the aid of 3·Methyl glucose 0 ·265 2 : 3 : 4 : 6-Tetramethyl galactose 0 ·88 a dry powder containing the bacterial Rhamnose 0 ·30 Tetramethyl fructopyranose 0·90 3 : 4-Dimethyl galactose 0·32 2 : 3 : 4-Trimethyl xylose 0·94 enzyme from Bacillus subtiliB, kindly 2-Methyl arabinose 0 ·36 2 : 3 : 5-Trimethyl arabinose 0 ·95 furnished by Prof. Linderstr"m-Lang, the 3 : 6-Anhydroglucose 0 ·37 2 : 3 : 4 : 6-Tetramethyl mannose 0·96 2-Methyl xhloee 0·385 2 : 3 : 4 : 6-Tetramethyl glucose 1·0 enzyme experiments were carried out as 2 : 4·Dimet yl galactose 0·405 2 : 3 : 4-Trlmethyl rhamnose 1·01 described by the Danish workers1• All the 2-Methyl fucose 0·51 o·o• 3 : 6-Dimethyl glucose 0·51 0·035 electrophoretic patterns were obtained after 4-Methyl rhamnose 0·575 P-Methyl arabinoside 0·325 electrophoresis of a 1·2 per cent solution 3 : 4-Dimethyl mannose 0·58 a-Methyl mannoslde 0·30 of the protein for four hours in a sodium phosphate buffer of pH 6·8 and 0·1 ionic • No movement. strength at a potential gradient of 6•5 volts per em. Under these conditions, The separation of the tetra.methyl derivatives of superposition of the patterns permits direct com• glucose, galactose and mannose is readily achieved. parison of the relative concentrations of the Furthermore, closely related pairs of substances, for components and indicates roughly their mobilities. example, 2 : 3-dimethyl xylose and 2 : 4-dimethyl• The patterns of the ascending boundaries are xylose, and 2 : 3 : 4-trimethyl galactose and 2 : 4 : 6- presented here because of better resolution. As a trimethyl galactose, are separable. The method result of adjustments occurring at the tl boundary, renders practicable a rapid examination of the peaks due to components having similar mobilities hydrolysis products obtained from small quantities will not coincide precisely. The mobilities cited below of methylated . It will facilitate also were therefore computed from the descending pat• the examination of fractions of methylated sugars terns. produced on distillation of the products of hydrolysis In Fig. 1 are superimposed the tracings of the pat• of methylated polysaccharides. The presence of terns of ovalbumin before and after incubation with methylated uronic acid derivatives causes tailing on the bacterial enzyme, and also after recrystallization, the paper chromatogram ; but we find that if these as plates, of the enzyme-treated material. In the are first removed with 'Amberlite Resin', IR4B, the case of the ovalbumin before and after enzyme treat• spots due to the various sugars may be readily ment, the qualitative appearance of the patterns is differentiated in the majority of cases. similar but the mobilities are different. Although By this method we have confirmed the presence of this difference is small, it is sufficient to lead one to the methylated sugar derivatives detected by the expect resolution of the two main components on distillation procedure in methylated cherry gum3, mixing the enzyme-treated protein and ovalbumin.

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