120. Paper Electrophoresis o f Hexosamines, N-Acetylhexosamines and N Acetylneuraminic Acid*l

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1 534 [Vol. 39, 120. Paper Electrophoresis o f Hexosamines, N-Acetylhexosamines and N Acetylneuraminic Acid*l By Seiichi OHKUMA and Toshiaki SHINOHARA Biochemical Laboratory, Scientific Police Research Institute, National Police Agency, Tokyo (Comm. by Tanemoto FURUHATA, M.J.A., Sept. 12, 1963) Paper electrophoresis with sodium tetraborate,l' potassium tetraborate,l''2' cetyltrimethylammonium borate2' or sodium molybdate3' buffer is a widely used procedure for the separation and identification of sugars. The paper electrophoresis with sodium tetraborate buffer has been also used for the separation and identification of 2-amino- 2-deoxy-aldoses and their N-acetyl derivatives by Crumpton,4' Maley & Maley5' and Jourdian & Rosemans' and these techniques have since found extensive applications. These methods suffer, however, from the disadvantage that 2-amino-2-deoxy-aldoses on papers buffered with sodium tetraborate cannot be detected with the Elson-Morgan spray reagents7' or other spray reagents.8' -12) The present paper describes color tests for the detection of hexosamines, N-acetylhexosamines and SA on borate-buffered papers and paper electrophoresis for the separation and identification of these sugars. Experimental. Buffers (Electrolytes). Potassium tetraborate, sodium tetraborate and sodium metaborate were examined as buffers. Paper electrophoresis. The instrument used was type C. for paper electrophoresis (Toyo Filter Paper Co., Tokyo. Japan). In each electrophoresis experiment, one paper strip (Whatman No. 3 MM. 10 cm wide X 34 cm long) was used. Before being placed in the electrophoresis apparatus, the strip was moistened with buffer and pressed between sheets of clean filter paper to remove the excess of buffer. The strip was then placed in the apparatus and sample solutions*2 were applied to the starting line*3 of 17 cm from the edge of the strip. The voltage was connected 5 min later, in order to allow the diffusion of the buffer. Electrophoresis was carried out by the hori- *1 The abbreviations used are: G1cN, Glucosamine; Ga1N, Galactosamine; ManN, Mannosamine; G1cNAc, N-Acetylglucosamine; GalNAc, N-Acetylgalactosamine; ManNAc, N-Acetylmannosamine; SA, N-Acetylneuraminic acid; M-3-AG, Methyl 3-amino-3-deoxya-D-glucopyranoside; M-6-AG, Methyl 6-amino-6-deoxy-a-D-glucopyranoside; dk, dark; 1, light; lg, a large spot; s, a small spot. *2 The amino sugar or N-acetylamino sugar solution in concentration of 5 mg/ml was used in this work. *3 The middle line of the strip.

2 No. 7] Paper Electrophoresis of Hexosamines, N-Acetylhexosamines etc. 535 zontal open strip method under a constant voltage. Platinium electrodes were used. Detection of spots on borate-buffered paper. Color test I (for the detection of 2-amino-2-deoxy-hexoses, 2-acetamino-2-deoxy-hexoses and SA):13' Immediately after electrophoresis, the paper strip was sprayed with the acetic anhydride-acetone spray reagent (this reagent was prepared freshly before use by mixing one part of acetic anhydride and four parts of acetone) and heated at 95 C for 5 min. The strip was then sprayed with the p-dimethylaminobenzaldehyde spray reagent7',13' and allowed to stand at room temperature. Color test II (for the detection of 2-acetamino-2-deoxy-hexoses and SA):13' Immediately after electrophoresis, the strip was heated at 95 C for 5 min and the strip was then sprayed with the p-dimethylaminobenzaldehyde spray reagent and allowed to stand at room temperature. Color test III (for the detection of M-3-AG, M-6-AG, * 4 kanamycin*5 and acid hydrolyzate of kanamycin*6) : Immediately after electrophoresis, the strip sprayed with the ninhydrin spray reagent (this reagent was prepared freshly before use by mixing nine parts of 0.2% solution of ninhydrin in n-butanol and one part of acetic acid) and heated at 105 C for 5 min. Results. Color tests. After electrophoresis at 200 V for 6 hr, when the papers were sprayed with the ninhydrin reagent, *7,7),8) aniline hydrogen phthalate,*7',7' benzidine-trichloroacetic acid reagent,9' aniline-diphenylamine-phosphoric acid reagent,10' alkaline silver nitrate reagent5~,7>,11>>12> and Elson-Morgan reagents 7' G1cN *4 GalN *4 ManN *4 G1cNAc, Ga1NAc, ManNAc and SA did not react. When the papers were treated under the conditions of color test I, the hexosamines and N-acetylhexosamines gave selectively violet spots and SA gave slowly a red violet spot against a white background. Therefore, color test I can be used for the selective detection of 2-amino-2-deoxyhexoses, 2-acetamino-2-deoxy-hexoses and SA on borate-buffered papers. After the same electrophoresis, when the papers were treated under the conditions of color test II, the N-acetylhexosamines gave violet spots and SA gave slowly a red violet spot against a white background. The hexosamines did not react under the same conditions. Color test II can be used for the specific detection of 2-acetamino-2-deoxy-hexoses and SA on borate-buffered papers. In color *4 The hydrochloride was used. *5 The sulfate was used. *S The acid hydrolyzate of kanamycin was obtained by hydrolysis of 30 mg of kanamycin.*5 with 5 ml of 2 N hydrochloric acid at 100 C for 20 hr in a sealed tube. *7 The reagent contains a small quantity of acetic acid.

3 536 S. OHKUMA and T. SHINOHARA [Vol. 39, tests I and II, the violet colors of the hexosamines and N-acetylhexosamines on papers were relatively unstable, while the red violet color of SA was stable for several days. Color test III may be adapted for the detection of 3-amino-3- deoxy-glucose, 6-amino-6-deoxy-glucose and their derivatives on boratebuffered papers and these amino sugars gave orange violet, brown violet or red violet spots against an almost white background. Under the conditions of color tests I and II, these amino sugars did not react. Paper electrophoresis of IV acetylhexosamines and SA. Influence of borate buffer, time and voltage on the electrophoretic mobilities and separations of the N-acetylhexosamines and SA were determined. 1. The influence of borate buffer. The electrophoretic mobilities of the N-acetylhexosamines and SA in potassium tetraborate, sodium tetraborate and sodium metaborate solutions of varying concentration are shown in Table I. In M potassium tetraborate, GalNAC, ManNAc and SA migrated to the anode, while G1cNAc migrated to the cathode. The almost same results were obtained in electrophoresis with M sodium tetraborate. In electrophoresis with M potassium tetraborate, a mixture of G1cNAc, GaINAc, ManNAc and SA gave four distinct violet spots as shown in Table II. These spots were identified as SA, ManNAc, GaINAc and G1cNAc, respectively, by their electrophoretic mobilities and colors. The results of electrophoresis of the same mixture with M sodium tetraborate were similar to those given by M potassium tetraborate, as shown in Table II, however the color intensities of these N-acetylhexosamines on paper electropherograms buffered with M potassium tetraborate were slightly stronger than those obtained by M sodium tetraborate. The electrophoretic mobilities of the N-acetylhexosamines Table I. Paper electrophoretic mobilities of N-acetylhexosamines and SA in different borate buffers of varying concentration (200 V, 6 hr. Migration distances in cm).

4 No. 7] Paper Electrophoresis of Hexosamines, N-Acetylhexosamines etc. 537 Table II. Paper electrophoretic separation of the N-acetylhexosamines and SA alone and in admixture (200 V, 6 hr) in potassium tetraborate and sodium tetraborate in concentration of each 0.05 M, were lower than those of each M. In sodium metaborate in concentration of M and 0.05 M, ManNAc migrated to the anode, while G1cNAc and GalNAC migrated to the cathode. In 0.1 M sodium metaborate, GalNAC, ManNAc and SA migrated to the anode and G1cNAc migrated to the cathode. In general, in electrophoresis with sodium metaborate, the N-acetylhexosamines gave faint or light violet spots and ManNAc gave a diffused spot. Therefore, it is clear that this borate buffer cannot be used for the separation and identification of the N-acetylhexosamines and SA. These results show that M potassium tetraborate or sodium tetraborate buffer is suitable for the paper electrophoresis of the N- acetylhexosamines and SA. 2. The influence of time. The influence of time on the electrophoretic mobilities of the N-acetylhexosamines was determined and the results were summarized in Table III. The results show that the mobility increases with an increase in the time of electrophoresis and that the most suitable time for electrophoresis is 6 hr. 3. The influence of voltage. The influence of voltage on the electrophoresic mobilities of the N-acetylhexosamines was determined and the results were summarized in Table IV. The results show that the mobility increases with an increse in the voltage of electrophoresis and that most suitable voltage for electrophoresis is 200 V. Paper electrophoresis of hexosamines and other amino sugars. When G1cN, Ga1N and ManN were subjected to electrophoresis with M potassium tetraborate and M sodium tetraborate, the hexosamines gave violet spots under the conditions of color test I. However, the color given by M potassium tetraborate tends to be a more intense violet than the color given by M sodium tetraborate. In electrophoresis with sodium metaborate in concentration of 0.025, 0.05 and 0.1 M, the hexosamines gave faint violet spots under the conditions of color test I. When G1cN, GalN, ManN and a mixture of G1cN, GalN and ManN were subjected to electrophoresis with M potassium tetraborate and M sodium tetraborate at 200 V for 6 hr, the results as shown in Table V were

5 538 S. OHKUMA and T. SHINOHARA [Vol. 39, Table III. Influence of time on the electrophoretic of the N-acetylhexosamines (200 V) mobilities Table IV. Influence of voltage on the electrophoretic of the N-acetylhexosamines (6 hr) mobilities Table V. Paper electrophoretic mobilities and separation of hexosamines alone and in admixture (200 V, 6 hr) the Table VI. Paper electrophoretic mobilities M potassium tetraborate of some amino (200V, 6 hr) sugars in obtained. It was observed in both tetraborates that GleN migrated to the cathode, GaiN and ManN migrated to the anode and that the mixture of the hexosamines was separated into two violet spots and these were : a violet spot, which was identified as ManN by its electrophoretic mobility, and other violet spot, which could not be identified as GIcN or GalN by its electrophoretic mobility. These results show that GleN and GalN cannot be separated from each other, but ManN can be completely separated from G1cN and GaiN under the conditions employed. When M-3-AG, M-6-AG, kanamycin and acid

6 No. 7] Paper Electrophoreisi of Hexosamines, N-Acetylhexosamines etc. 539 hydrolyzate of kanamycin were subjected to the electrophoresis with M potassium tetraborate at 200 V for 6 hr, the results as shown in Table VI were obtained. Conclusions. From these studies, the following conclusions were deduced : (1) Color tests I, II and III are useful for the detection of 2- amino-2-deoxy-hexoses, 2-acetamino-2-deoxy-hexoses, N-acetylneuraminic acid and other amino sugars on papers buffered with borate solutions and these classes can be distinguished on paper electropherograms through the manipulation of the color tests I, II and III. (2) The separation and identification of 2-acetamino-2-deoxyhexoses and N-acetylneuraminic acid in admixture, can be satisfactorily accomplished by paper electrophoresis with M potassium tetraborate or M sodium tetraborate at 200 V for 6 hr. (3) The electrophoresic method described at (2) can also be used as a tool for the identification of 2-amino-2-deoxy-hexose alone, but cannot be applied to the separation and identification of the hexosamines in admixture. Acknowledgement. The authors wish to thank Prof. T. Furuhata, of the director of this institute for his guidance and to thank Dr. M. Watanabe and Dr. S. Watanabe for their support in this work. Summary. Color tests for the detection of hexosamines, N-acetylhexosamines, N-acetylneuraminic acid and other amino sugars on papers buffered with borate solutions are described. A method is described which permits the separation and identification of these sugars by paper electrophoresis. 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) References Foster, A. B.: Advances in Carbohydrate Chemistry, 12, 81 (1957). Piras, R., and Cabib, E.: J. Chromatog., 8, 63 (1962). Bourne, E. J., Hutson, D. H., and Weigel, H.: Chem. & Ind., 1959, 1047; Anal. Abst., 7, 1787 (1960). Crumpton, M. J.: Biochem. J., 72, 479 (1959). Jourdian, G. W., and Roseman, S.: J. Biol. Chem., 237, 2442 (1962). Maley, F., and Maley, G. F.: Biochem. Biophys. Acta., 31, 577 (1959). Partridge, S. M.: Biochem. J., 42, 238 (1948). Aminofi, D., and Morgan, W. T. J.: Nature, 162, 579 (1948). Bacon, J. S. D., and Edelman, J.: Biochem. J., 48, 114, (1951). Buchan, J. L., and Savage, R. I.: Analyst., 77, 401 (1952). Partridge, S. M.: Nature. 158, 270 (1946). Trelevyan, W. E., Procter, D. P., and Harrison, J. S.: Nature, 166, 444 (1950). Ohkuma, S.: Proc. Japan. Acad., 39, 400 (1963).

774 [Vol. 39, *) The abbreviations used are: GIcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine;

774 [Vol. 39, *) The abbreviations used are: GIcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; 774 [Vol. 39, 170. Separation and Identification of N-Acetylhexosamines and N Acetylneuraminic Acid by Two-dimensional Electrophoresis and Chromatography on Paper By Seiichi OHKUMA and Toshiaki SHINOHARA