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A method for quantitative recovery of acidic glycosaminoglycans after electrophoresis on cellulose acetate strip
ANALYTICAL
BlOCHEMISTRY
65. 537-539
SHORT
(1975)
COMMUNICATIONS
A Method for Quantitative Recovery of Acidic Glycosaminoglycans After Electrophoresis on Cellulose
Acetate
Strip
Acidic glycosaminoglycans after electrophoretic separation on a strip of cellulose acetate membrane were stained with toluidine blue or azure I. The basic dye was removed with 0.2 M NH,OH in 80% ethyl alcohol. Then elution with water was effected for quantitative recovery of glycosaminoglycans.
Electrophoresis on cellulose acetate membrane has been used for the separation of acidic glycosaminoglycans (l-8). Their quantitation has been achieved by calorimetric evaluation of the electrophoretogram stained with basic dyes (3-5) or by quantitative estimation of the dyes dissociated from the glycosaminoglycan-dye complexes (8). It would be of advantage if glycosaminoglycans free from dyes could be recovered quantitatively. This paper deals with a method for the quantitative recovery of glycosaminoglycans from cellulose acetate strips after removal of the dyes from the glycosaminoglycan-dye spots. Materids. Hyaluronic acid and dermatan sulfate were prepared from human umbilical cord according to the procedure of Danishefsky and Bella (9). Chondroitin 4-sulfate, chondroitin 6-sulfate, and keratan sulfate were purchased from Seikagaku Kogyo Co. Ltd., Tokyo. Heparan sulfate was a generous gift from Dr. M. B. Mathews, University of Chicago. Bovine heparin was purchased from Taiyo Fishery Co. Ltd., Yokosuka. These materials after purification gave a homogeneous spot in electrophoresis on cellulose acetate strips in more than two solvent systems. Toluidine blue (E. Merck AG, Darmstadt), methylene blue (Wako Pure Chemical Industries Ltd., Osaka), azure I (Koso Chemical Co. Ltd., Tokyo), and alcian blue EGS (Chroma-Gesellschaft Schmid & Co., Stuttgart-Unterttirkheim) were used for staining. All the reagents used were of analytical grade. Separax (cellulose acetate membrane) was purchased from Joko Sangyo Co. Ltd., Tokyo. Determination of constituents. The constituents of glycosaminoglycans were analyzed by the following methods: hexosamine by the Elson-Morgan reaction (10) and/or by the indole-HCl reaction (11,12); hexose by the phenol-H,SO, method (13); uranic acid by the modified 537 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form resewed.
538
SHORT COMMUNICATIONS
carbazole reaction (14); sulfate by the benzidine method of Antonopoulos (15). Elrctrophoresis. An aqueous solution of glycosaminoglycans ( lo- 100 pg in lo-50 ~1) was applied on a strip (6.5 X 10 cm) of Separax. Electrophoresis was carried out in 0.1 M formic acid-pyridine buffer (pH 3.0) at 0.5 mA/cm for 40 min. The glycosaminoglycans on electrophoretogram were located by staining with toluidine blue or azure I (0.05% in 80% ethyl alcohol). Excess dye was removed by rinsing with 80% ethyl alcohol. The strip was then blotted on a filter paper. The glycosaminoglycan-dye spot was cut into small pieces, and put into a test tube with a glass-joint stopper. The glycosaminoglycan-dye complex was dissociated with 3 ml of 0.2 M NH,OH in 80% ethyl alcohol with occasional shakings at room temperature. This procedure of dissociation was repeated several times using fresh dissociation medium until the pieces became colorless. Finally, the decolorized pieces were rinsed with 80% ethyl alcohol. After removal of the dye as described above, the glycosaminoglycan on cellulose acetate was eluted with water overnight at room temperature. After centrifugation, the pieces were washed with water. The resulting supernatant and the washing were combined and concentrated to dryness. Results and discussion. One of the representative data of the recoveries of glycosaminoglycans is listed in Table I. Similar recoveries were observed with lo-100 pug of acidic glycosaminoglycans. All the electrophoretic methods reported previously for the separation of glycosaminoglycans (1-8) are applicable. It was found that 0.2 M NH,OH in 80% ethyl alcohol was the best of the media thus far tested for the removal of the dye from the glycosaminoglycan-dye complexes on TABLE
1
PERCENT RECOVERIES OF ACIDIC GLYCOSAMINOGLYCANS AFTER THE ELIMINATION DYE FROM THE GLYCOSAMINOGLYCAN-DYE SPOTS OBTAINED BY ELECTROPHORESIS CELLULOSE ACETATE MEMBRANE IN 0.1 M FORMIC ACID-PYRIDINE BUFFER (pH AT 0.5 mA/cm FOR 40 MIN. STAINING WITH TOLUIDINE BLUE (0.05% IN 80% ETHYL ALCOHOL)
Hyaluronic acid Chondroitin 4-sulfate Chondroitin 6-sulfate Dermatan sulfate Heparin Heparan sulfate Keratan sulfate
Hexosamine
Uranic acid
94.0
97.2 96.0 92.4 94.3 93.6 92.1
96.1 94.3
92.1 92.7
94.5 88.1
Q Expressed as percentage of recoveries materials.
Hexose
OF ON 3.0)
Sulfate 92.9
91.1
90.0
90.4 91.5 90.3 87.7
of the constituents compared to the original
SHORT COMMUNICATIONS
539
cellulose acetate pieces. Under the present condition, 0.2 M NH40H was strong enough in alkalinity to dissociate the glycosaminoglycandye complexes, and the released dye was easily dissolved in 80% ethyl alcohol. The giy~osaminogiy~ans recovered showed the same mobilities in electrophoresis and analytical data as the giycosaminoglycans before eiectrophoresis. Thus, no noticeable change was found in glycosaminoglycans after eiectrophoresis. Toiuidine blue, methyiene blue, and azure I can be used for staining of giycosaminogiycans, but aician blue is not applicable, because aician blue is converted into a stable “~onastrai fast blue” in an alkaline solution (16). The above results show that the present technique makes it possible to isolate glycosaminoglycans in high purity on a microscale without any noticeable change of their polysaccharide chains. ACKNOWLEDGMENTS We thank Dr. M. B. Mathews for heparan sulfate. This work was supported by a grant from the Ministry of Education of Japan.
REFERENCES 1. Mathews, M. B. (1961) B~[~~~~it?z. Biuphp. Acra 48, 402-403. Wessler, E. (1968) Anal. ~j(~~,~?~~~r. 26, 439-444. 3. Kimura, A., and Tsurumi, K. (I 969) J. Biochem. 65, 303-304. 4. Prout, R. E. S. (1969) Biochim. Biophys. Artcr 177, 157-158. 5. Seno. N.. Anno, K., Kondo, K., Nagase, S., and Saito, S. (1970) Anal. Biochem. 37, 197-202. 6. Wessler, E. f 1971) And. Biod~cvn. 41, 67-69. 7. Hata, R., and Nagai, Y. (1972) Anal. Bj~~~~ern. 45, 462-468. 8. Hata, R., and Nagai, Y. f 1973) Anal. Bj(~~,~?e~~. 52, 652-656. 9. Danishefsky, I., and Bella, A., Jr. (1966) 1. Bid. Chem. 241, 143-146. 10. Blix, G. (1948) Acta Chrm. Scand. 2, 467-473. 11. Dische, Z., and Borenfreund, E. ( 1950) J. Bid. Cham. 184, 5 17-522. 12. Yosizawa, Z. (1961) Tohokr J. Exp. Med. 74, 69-75. 13. Dubois, M., Titles, K. A., Hamilton, J. K.. Rebers. P. A., and Smith, F. (1956) A&. 2.
Chem.
28, 350-356.
14. Bitter, T.. and Muir, H. M. (1962) Amt. Bi~~~~lern. 4, 330-334. IS. Antonopouios, C. A. (1962) Ada Chew. Swnd. 16, 1521-1522. 16. Movat. H. Z. (1955) A MA Arch. Patho/. 57, 289-395.
BlOCHEMISTRY
65. 537-539
SHORT
(1975)
COMMUNICATIONS
A Method for Quantitative Recovery of Acidic Glycosaminoglycans After Electrophoresis on Cellulose
Acetate
Strip
Acidic glycosaminoglycans after electrophoretic separation on a strip of cellulose acetate membrane were stained with toluidine blue or azure I. The basic dye was removed with 0.2 M NH,OH in 80% ethyl alcohol. Then elution with water was effected for quantitative recovery of glycosaminoglycans.
Electrophoresis on cellulose acetate membrane has been used for the separation of acidic glycosaminoglycans (l-8). Their quantitation has been achieved by calorimetric evaluation of the electrophoretogram stained with basic dyes (3-5) or by quantitative estimation of the dyes dissociated from the glycosaminoglycan-dye complexes (8). It would be of advantage if glycosaminoglycans free from dyes could be recovered quantitatively. This paper deals with a method for the quantitative recovery of glycosaminoglycans from cellulose acetate strips after removal of the dyes from the glycosaminoglycan-dye spots. Materids. Hyaluronic acid and dermatan sulfate were prepared from human umbilical cord according to the procedure of Danishefsky and Bella (9). Chondroitin 4-sulfate, chondroitin 6-sulfate, and keratan sulfate were purchased from Seikagaku Kogyo Co. Ltd., Tokyo. Heparan sulfate was a generous gift from Dr. M. B. Mathews, University of Chicago. Bovine heparin was purchased from Taiyo Fishery Co. Ltd., Yokosuka. These materials after purification gave a homogeneous spot in electrophoresis on cellulose acetate strips in more than two solvent systems. Toluidine blue (E. Merck AG, Darmstadt), methylene blue (Wako Pure Chemical Industries Ltd., Osaka), azure I (Koso Chemical Co. Ltd., Tokyo), and alcian blue EGS (Chroma-Gesellschaft Schmid & Co., Stuttgart-Unterttirkheim) were used for staining. All the reagents used were of analytical grade. Separax (cellulose acetate membrane) was purchased from Joko Sangyo Co. Ltd., Tokyo. Determination of constituents. The constituents of glycosaminoglycans were analyzed by the following methods: hexosamine by the Elson-Morgan reaction (10) and/or by the indole-HCl reaction (11,12); hexose by the phenol-H,SO, method (13); uranic acid by the modified 537 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form resewed.
538
SHORT COMMUNICATIONS
carbazole reaction (14); sulfate by the benzidine method of Antonopoulos (15). Elrctrophoresis. An aqueous solution of glycosaminoglycans ( lo- 100 pg in lo-50 ~1) was applied on a strip (6.5 X 10 cm) of Separax. Electrophoresis was carried out in 0.1 M formic acid-pyridine buffer (pH 3.0) at 0.5 mA/cm for 40 min. The glycosaminoglycans on electrophoretogram were located by staining with toluidine blue or azure I (0.05% in 80% ethyl alcohol). Excess dye was removed by rinsing with 80% ethyl alcohol. The strip was then blotted on a filter paper. The glycosaminoglycan-dye spot was cut into small pieces, and put into a test tube with a glass-joint stopper. The glycosaminoglycan-dye complex was dissociated with 3 ml of 0.2 M NH,OH in 80% ethyl alcohol with occasional shakings at room temperature. This procedure of dissociation was repeated several times using fresh dissociation medium until the pieces became colorless. Finally, the decolorized pieces were rinsed with 80% ethyl alcohol. After removal of the dye as described above, the glycosaminoglycan on cellulose acetate was eluted with water overnight at room temperature. After centrifugation, the pieces were washed with water. The resulting supernatant and the washing were combined and concentrated to dryness. Results and discussion. One of the representative data of the recoveries of glycosaminoglycans is listed in Table I. Similar recoveries were observed with lo-100 pug of acidic glycosaminoglycans. All the electrophoretic methods reported previously for the separation of glycosaminoglycans (1-8) are applicable. It was found that 0.2 M NH,OH in 80% ethyl alcohol was the best of the media thus far tested for the removal of the dye from the glycosaminoglycan-dye complexes on TABLE
1
PERCENT RECOVERIES OF ACIDIC GLYCOSAMINOGLYCANS AFTER THE ELIMINATION DYE FROM THE GLYCOSAMINOGLYCAN-DYE SPOTS OBTAINED BY ELECTROPHORESIS CELLULOSE ACETATE MEMBRANE IN 0.1 M FORMIC ACID-PYRIDINE BUFFER (pH AT 0.5 mA/cm FOR 40 MIN. STAINING WITH TOLUIDINE BLUE (0.05% IN 80% ETHYL ALCOHOL)
Hyaluronic acid Chondroitin 4-sulfate Chondroitin 6-sulfate Dermatan sulfate Heparin Heparan sulfate Keratan sulfate
Hexosamine
Uranic acid
94.0
97.2 96.0 92.4 94.3 93.6 92.1
96.1 94.3
92.1 92.7
94.5 88.1
Q Expressed as percentage of recoveries materials.
Hexose
OF ON 3.0)
Sulfate 92.9
91.1
90.0
90.4 91.5 90.3 87.7
of the constituents compared to the original
SHORT COMMUNICATIONS
539
cellulose acetate pieces. Under the present condition, 0.2 M NH40H was strong enough in alkalinity to dissociate the glycosaminoglycandye complexes, and the released dye was easily dissolved in 80% ethyl alcohol. The giy~osaminogiy~ans recovered showed the same mobilities in electrophoresis and analytical data as the giycosaminoglycans before eiectrophoresis. Thus, no noticeable change was found in glycosaminoglycans after eiectrophoresis. Toiuidine blue, methyiene blue, and azure I can be used for staining of giycosaminogiycans, but aician blue is not applicable, because aician blue is converted into a stable “~onastrai fast blue” in an alkaline solution (16). The above results show that the present technique makes it possible to isolate glycosaminoglycans in high purity on a microscale without any noticeable change of their polysaccharide chains. ACKNOWLEDGMENTS We thank Dr. M. B. Mathews for heparan sulfate. This work was supported by a grant from the Ministry of Education of Japan.
REFERENCES 1. Mathews, M. B. (1961) B~[~~~~it?z. Biuphp. Acra 48, 402-403. Wessler, E. (1968) Anal. ~j(~~,~?~~~r. 26, 439-444. 3. Kimura, A., and Tsurumi, K. (I 969) J. Biochem. 65, 303-304. 4. Prout, R. E. S. (1969) Biochim. Biophys. Artcr 177, 157-158. 5. Seno. N.. Anno, K., Kondo, K., Nagase, S., and Saito, S. (1970) Anal. Biochem. 37, 197-202. 6. Wessler, E. f 1971) And. Biod~cvn. 41, 67-69. 7. Hata, R., and Nagai, Y. (1972) Anal. Bj~~~~ern. 45, 462-468. 8. Hata, R., and Nagai, Y. f 1973) Anal. Bj(~~,~?e~~. 52, 652-656. 9. Danishefsky, I., and Bella, A., Jr. (1966) 1. Bid. Chem. 241, 143-146. 10. Blix, G. (1948) Acta Chrm. Scand. 2, 467-473. 11. Dische, Z., and Borenfreund, E. ( 1950) J. Bid. Cham. 184, 5 17-522. 12. Yosizawa, Z. (1961) Tohokr J. Exp. Med. 74, 69-75. 13. Dubois, M., Titles, K. A., Hamilton, J. K.. Rebers. P. A., and Smith, F. (1956) A&. 2.
Chem.
28, 350-356.
14. Bitter, T.. and Muir, H. M. (1962) Amt. Bi~~~~lern. 4, 330-334. IS. Antonopouios, C. A. (1962) Ada Chew. Swnd. 16, 1521-1522. 16. Movat. H. Z. (1955) A MA Arch. Patho/. 57, 289-395.