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Electrophoresis of acidic glycosaminoglycans in hydrochloric acid: A micro method for sulfate determination
.,S.ILTTICAL
BIOCHE>118THT
41, 67-60 (1971)
Electrophoresis in A Micro
of Acidic Hydrochloric
Method
Glycosaminoglycans Acid:
for Sulfate
Received Srptemh
Determination
2, 1970
Acidic glycosaminoglycans may by divided into several groups according to the structure of their polysaccharide backbone (1). Each group is heterogeneous with respect to molecular weight and, in most cases, with respect to charge density. The charge polydispersity reflects variations in the degree of sulfation. Current methods for determination of sulfate in acidic glycosaminoglycans arc laborious and require relatively large amounts of material. The present communication describes a simple micro method for estimating the sulfate content of glycosaminoglycans. MATERIALS
ASD
METHODS
Glycosaminoglycans. Hyaluronic acid from human umbilical cord was a gift from Dr. G. Blix of this institute. Chondroitin sulfate, heparan sulfate, and keratan sulfate fractions with varying sulfate content were prepared from normal human urine by means of proteolytic digestion and chromatography on ECTEOLA-cellulose, followed by preparative electrophoresis in solution barbital buffer and barium acetate (2j. The heparin used, a commercial preparation from pig intestinal mucosa, was further purified by precipitation with cetylpyridinium chloride from 1.2 M sodium chloride and was kindly provided by Dr. U. Lindahl of this institute. Hexosamine was assayed by the ElsonMorgan procedure (3) as described previously (2). Sulfate was determined by the benzidine method of Antonopoulos (4). Electlophoresis was performed in 0.1 N hydrochloric acid on strips, 4 X 30 cm, of cellulose acetate (Membranfiltergesellschaft GmhH, G6ttingen, Germany). The suspdca and immersed (Varsol) strip methods were used (5). Samples of polysaccharidc (about 0.2 pg in 0.2 ~1) were applied to the middle of the strips and were then exposed to a potential gradient of 2.5 V/cm for 3 hr. Staining was performed with Alcian blue as described earlier (6). 67
RESULTS
AND
DISCUSSlON
Since the carboxyl groups of uranic acids are undissociatecl in 0.1 M hydrochloric acid (pH about 1.2) (7)) the charge density of glycosaminoglycans in this medium should reflect their sulfate content. Accordingly, on electrophoresis under these conditions, heparin migrates faster than whereas the nonsulfated polythe monosulfated glycosaminoglycans, saccharide, hyaluronic acid, remains at the origin (8). In contrast to electrophoresis in other media (6)) their mobilities in 0.1 M hydrochloric acid do not depend on the structure of the polysaccharide backbone, as preparations of chondroitin sulfate, dermatan sulfate, and heparan sulfate with similar sulfate contents have the same mobility (8). The relation between electrophoretic mobility and sulfate content was investigated further by subjecting a number of glycosaminoglycan preparations of varying sulfate contents to electrophoresis in 0.1 M hydrochloric acid. The results (Fig. 1) show a linear relationship between the parameters with a correlation coefficient of 0.95. Thus, the sulfate content of a polysaccharide may be estimated by comparing the migration rate with that of a single standard. In the experiment shown in Fig. 1, the strips were immersed in Varsol to avoid buffer flow due to evaporation. Wit,h the suspended-strip method, the relation between sulfate cont’ent and migration distance is nonlinear and standards with
Molar
rat/o
sulfatethexosam~ne
FIG. 1. Electrophoretic mobility of glycosaminoglycans in 0.1 A4 hydrochloric acid plotted against sulfate content: (0) hyaluronic acid; (0) chondroitin sulfate fractions; (0) heparan sulfate fractions; ( n ) heparin; (A) keratan sulfate fractions. Most of the sulfate fractions produced elongated spots. The position of the center of each spot is indicated.
SULFATE
ASSAY
IN
69
GLYCOSAMIXOGLYCAKS
known sulfate content must be used. The method cannot be used in the presence of proteins as they interact with the polysaccharides. The method has several applications: the degree of sulfation of a polysaccharide can be determined rapidly using less than 1 pg; and small differences in sulfate content can be detected. A polysaccharide fraction with a molar ratio of sulfate to hexosamine of 0.1 is readily differentiated from a nonsulfated fraction (2). Presumably, the appearance of elongated spots is partly due to the presence of chains differing in sulfate content, and thereby demonstrates charge heterogeneity within a single polysaccharide fraction. The distribution of the sulfate residues along the polysaccharide chain has yet to be established. Electrophoresis in 0.1-M hydrochloric acid of a chondroitin 4-sulfate fraction before and after limited endopolysaccharidase degration suggested differences in sulfate density in different parts of the same polysaccharide chain (9). SUMMARY
A rapid and simple m&hod for estimation of the sulfat,e content of less than 1 pg of acidic glycosaminoglycans is presented. The procedure is based on the fact that the electrophoretic mobility in 0.1 M hydrochloric acid is proportional to the sulfate content of the polysaccharide. ACKNOWLEDGMENTS The excellent technical assist,ance of Mrs. Elwy Andersson is gratefully acknowledged. This work was supported by grants from Konung the Medical Faculty, University of Uppsala.
WaIlin
and Miss
Gunilla
Gustaf V:s 89-&rsfond
and
REFERENCES R. W.. i)r “Comprehensive Biochemistry” (Florkin, M., and Stotz, E. H., eds.), Vol. 5, p. 262. Elsevier, Amsterdam, 1963. 2. WESSLER, E.. &o&em. J., in press. 3. ELSON, L. A., AND MORGAN, W. T. J.. BiochenL. J. 27, 1824 (1933). 4. ANTONOPOULQS, (;“. ,4., Acta Chem. Scn~d. 16, 1521 (1962). 5. zWEIc3, G., AND WHITAKER, J. R., “Paper Chromatography and Electrophoresis.” Vol. 1, ‘LElectrophoresis in Stablizing Media” (by J. R. Whitaker). Academic Press, New York, 1967. 6. WESSLER, E., Anal. Biochem. 26, 439 (1968). 7. MATHEWS, M. B., Biochem. Biophys. Actn 48, 402 (1961). 8. WESSLER, E., in “The Chemist.ry a.nd Molecular Biology of the Intercellular Matrix” (Balsas, E. A.? rd.). p. S95. Academic Press, London/New York, 1979. 9. WASTESON~ x.. AND IJIN~.411L. IT.. submitted for publication to J. Biol. Chem. 1. JEANLOZ.
BIOCHE>118THT
41, 67-60 (1971)
Electrophoresis in A Micro
of Acidic Hydrochloric
Method
Glycosaminoglycans Acid:
for Sulfate
Received Srptemh
Determination
2, 1970
Acidic glycosaminoglycans may by divided into several groups according to the structure of their polysaccharide backbone (1). Each group is heterogeneous with respect to molecular weight and, in most cases, with respect to charge density. The charge polydispersity reflects variations in the degree of sulfation. Current methods for determination of sulfate in acidic glycosaminoglycans arc laborious and require relatively large amounts of material. The present communication describes a simple micro method for estimating the sulfate content of glycosaminoglycans. MATERIALS
ASD
METHODS
Glycosaminoglycans. Hyaluronic acid from human umbilical cord was a gift from Dr. G. Blix of this institute. Chondroitin sulfate, heparan sulfate, and keratan sulfate fractions with varying sulfate content were prepared from normal human urine by means of proteolytic digestion and chromatography on ECTEOLA-cellulose, followed by preparative electrophoresis in solution barbital buffer and barium acetate (2j. The heparin used, a commercial preparation from pig intestinal mucosa, was further purified by precipitation with cetylpyridinium chloride from 1.2 M sodium chloride and was kindly provided by Dr. U. Lindahl of this institute. Hexosamine was assayed by the ElsonMorgan procedure (3) as described previously (2). Sulfate was determined by the benzidine method of Antonopoulos (4). Electlophoresis was performed in 0.1 N hydrochloric acid on strips, 4 X 30 cm, of cellulose acetate (Membranfiltergesellschaft GmhH, G6ttingen, Germany). The suspdca and immersed (Varsol) strip methods were used (5). Samples of polysaccharidc (about 0.2 pg in 0.2 ~1) were applied to the middle of the strips and were then exposed to a potential gradient of 2.5 V/cm for 3 hr. Staining was performed with Alcian blue as described earlier (6). 67
RESULTS
AND
DISCUSSlON
Since the carboxyl groups of uranic acids are undissociatecl in 0.1 M hydrochloric acid (pH about 1.2) (7)) the charge density of glycosaminoglycans in this medium should reflect their sulfate content. Accordingly, on electrophoresis under these conditions, heparin migrates faster than whereas the nonsulfated polythe monosulfated glycosaminoglycans, saccharide, hyaluronic acid, remains at the origin (8). In contrast to electrophoresis in other media (6)) their mobilities in 0.1 M hydrochloric acid do not depend on the structure of the polysaccharide backbone, as preparations of chondroitin sulfate, dermatan sulfate, and heparan sulfate with similar sulfate contents have the same mobility (8). The relation between electrophoretic mobility and sulfate content was investigated further by subjecting a number of glycosaminoglycan preparations of varying sulfate contents to electrophoresis in 0.1 M hydrochloric acid. The results (Fig. 1) show a linear relationship between the parameters with a correlation coefficient of 0.95. Thus, the sulfate content of a polysaccharide may be estimated by comparing the migration rate with that of a single standard. In the experiment shown in Fig. 1, the strips were immersed in Varsol to avoid buffer flow due to evaporation. Wit,h the suspended-strip method, the relation between sulfate cont’ent and migration distance is nonlinear and standards with
Molar
rat/o
sulfatethexosam~ne
FIG. 1. Electrophoretic mobility of glycosaminoglycans in 0.1 A4 hydrochloric acid plotted against sulfate content: (0) hyaluronic acid; (0) chondroitin sulfate fractions; (0) heparan sulfate fractions; ( n ) heparin; (A) keratan sulfate fractions. Most of the sulfate fractions produced elongated spots. The position of the center of each spot is indicated.
SULFATE
ASSAY
IN
69
GLYCOSAMIXOGLYCAKS
known sulfate content must be used. The method cannot be used in the presence of proteins as they interact with the polysaccharides. The method has several applications: the degree of sulfation of a polysaccharide can be determined rapidly using less than 1 pg; and small differences in sulfate content can be detected. A polysaccharide fraction with a molar ratio of sulfate to hexosamine of 0.1 is readily differentiated from a nonsulfated fraction (2). Presumably, the appearance of elongated spots is partly due to the presence of chains differing in sulfate content, and thereby demonstrates charge heterogeneity within a single polysaccharide fraction. The distribution of the sulfate residues along the polysaccharide chain has yet to be established. Electrophoresis in 0.1-M hydrochloric acid of a chondroitin 4-sulfate fraction before and after limited endopolysaccharidase degration suggested differences in sulfate density in different parts of the same polysaccharide chain (9). SUMMARY
A rapid and simple m&hod for estimation of the sulfat,e content of less than 1 pg of acidic glycosaminoglycans is presented. The procedure is based on the fact that the electrophoretic mobility in 0.1 M hydrochloric acid is proportional to the sulfate content of the polysaccharide. ACKNOWLEDGMENTS The excellent technical assist,ance of Mrs. Elwy Andersson is gratefully acknowledged. This work was supported by grants from Konung the Medical Faculty, University of Uppsala.
WaIlin
and Miss
Gunilla
Gustaf V:s 89-&rsfond
and
REFERENCES R. W.. i)r “Comprehensive Biochemistry” (Florkin, M., and Stotz, E. H., eds.), Vol. 5, p. 262. Elsevier, Amsterdam, 1963. 2. WESSLER, E.. &o&em. J., in press. 3. ELSON, L. A., AND MORGAN, W. T. J.. BiochenL. J. 27, 1824 (1933). 4. ANTONOPOULQS, (;“. ,4., Acta Chem. Scn~d. 16, 1521 (1962). 5. zWEIc3, G., AND WHITAKER, J. R., “Paper Chromatography and Electrophoresis.” Vol. 1, ‘LElectrophoresis in Stablizing Media” (by J. R. Whitaker). Academic Press, New York, 1967. 6. WESSLER, E., Anal. Biochem. 26, 439 (1968). 7. MATHEWS, M. B., Biochem. Biophys. Actn 48, 402 (1961). 8. WESSLER, E., in “The Chemist.ry a.nd Molecular Biology of the Intercellular Matrix” (Balsas, E. A.? rd.). p. S95. Academic Press, London/New York, 1979. 9. WASTESON~ x.. AND IJIN~.411L. IT.. submitted for publication to J. Biol. Chem. 1. JEANLOZ.