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Gel electrophoresis of proteoglycans and glycosaminoglycans on large-pore composite polyacrylamide-agarose gels
ANALYTICAL
BIOCHEMISTRY
Gel
44,
Electrophoresis aminoglycans
612-622 (1971)
of Proteoglycans on
Large-Pore
Polyacrylamide-Agarose C. A. McDEVITT Kennedy
Institute
AND HELEN
and
Glycos-
Composite Gels MUIR
of Rheumatology, Bute Gardens, Hammersmith, London, W. 6, United Kingdom
Received May 5, 1971; revised July 2, 1971
The heterogeneity of proteoglycans of cartilage is now well established (l-5). Articular cartilage contains populations of proteoglycans of different size, the relative proportions of which vary with the age and pathological state of the tissue (6-8). The large Stokes radii (9,lO) of these hydrated molecules have made it difficult to apply sensitive highresolution separation techniques in the study of their heterogeneity. Although glycosaminoglycans (“acidic mucopolysaccharides”) have been separated in 6% polyacrylamide gels (11)) the intact proteoglycans are completely immobilized in 7% gels (12). Polyacrylamide gel electrophoresis has not been applied before to proteoglycans. Pore size is inversely related to the concentration of acrylamide (13) but polyacrylamide does not form gels below concentrations of 2% (14). Peacock and Dingman (15) have, however, succeeded in forming 1.5% polyacrylamide gels to separate high molecular weight RNA mixtures by incorporating 0.5% agarose into the gel medium for support. By modifying the technique of Peacock and Dingman, we have been able to form stable gels, comprised of 0.5-1.50/o acrylamide and 0.6% agarose, which are sufficiently porous to enable all but the very largest proteoglycans to penetrate them. Glycosaminoglycans migrate on these gels as sharp bands, the mobilities of which are a function of their charge density, whereas proteoglycans which are of similar charge density display mobilities that are inversely related to their molecular size. METHODS
Gel Preparation Acrylamide and N,N’-methylenebisacrylamide were recrystallized from chloroform and acetone, respectively, by the method of Loening 612
GEL
ELECTROPHORESIS
OF
PROTEOGLYCANS
613
(16). The buffer used was 0.04 1M Tris-acetate containing 1 mM sodium sulfate (final concentrations) adjusted at 4°C to pH 6.8 with acetic acid. Tris-acetate was used instead of the chloride to avoid formation of hypochlorite by electrolysis (16). The presence of sodium sulfate in the buffer prevented adsorption of the glyeosaminoglycans on to the composite gel matrix which would otherwise occur. The gels consisted in the main of 1.2% acrylamide and 0.6% agarose and were prepared as follows: Agarose, 0.48 gm (British Drug Houses, Ltd, Poole, Dorset, England, ‘
614
MCDEVITT
AND
MUIR
gels were then carefully replaced in the Perspex tubes, which were resealed at one end with Visking tubing and Parafilm. Electrophoretic
Conditions
The electrode buffer consisted of the Tris buffer diluted four times, to which EDTA was added to a concentration of 1 m.M to eliminate the possibility of heavy met,als affecting the electrophoresis of the proteoglycans (16). When electrophoresis was performed at low pH, the electrode buffer was a 1: 10 dilution of the 0.02 M citrate phosphate buffer and EDTA was again added to a concentration of 1 mM. The volume of the anode buffer was 400 ml and the volume of the cathode buffer 250 ml. The Perspex tubes with the Parafilm removed were placed vertically in the electrophoresis chamber (Pleuger S. A., Winjnegem, Belgium), and to ensure adequate cooling they were immersed up to the top of the gels in the buffer of the anode compartment. A current of 4 mA/tube (200 V) was applied for 1 hr to remove unpolymerized acrylamide and catalysts from the gels. Fresh buffer was then poured into the electrode compartments and samples of proteoglycans (2-20 pg) in 20 all of a 40% sucrose-water solution were layered on to the top of the gels. A voltage gradient of 13.8 V/cm was applied until a bromophenol blue marker entered the gel. The current was then increased to 4 mA/tube for the duration of the run (about 60 min) . The voltage gradient was about 27.8 V/cm. The temperature of both electrode buffers was 7.6”C before electrophoresis. The temperature of the cathode buffer rose to 11.5” and that of the anode buffer remained constant after 1 hr of electrophoresis. It was essential that electrophoresis was performed in the cold room and that the current did not rise above 5 mA/tube. Localization
of Bands
The gels were removed from their Perspex tubes and stained for 15 min in 0.2% toluidine blue in 0.1 N acetic acid. This dye forms waterinsoluble metachromatic complexes with proteoglycans (18). The excess dye was removed by immersing the gels in 3% acetic acid for 90 min, followed by 34 repeated washes in water and then overnight in water. The gels were then scanned with a Locarte gel densitometer using a filter allowing maximum transmission at 520 nm (Ilford filter No. 624). Neither Alcian blue nor acridine orange was satisfactory because they stained the composite gel matrix too strongly and were not removed during destaining. The distance from the base of the meniscus at the top of the gel to the point of maximum intensity of each band was measured in millimeters from the densitometric scans. A sample of articular cartilage chondroitin sulfate was included in all
GEL ELECTROPHORESIS
OF PROTEOGLYCAhTS
runs as a reference standard. The relative mobility expressed as a percentage of the distance migrated fate under the same conditions.
615
of each band was by chondroitin sul-
Source of Proteoglycans and Glycosaminoglycans
A comparative study of the mobilities of chondroitin sulfate, heparan sulfate, and hyaluronic acid was undertaken to assess the affect of charge density on the mobilities of glycosaminoglycans on the composite gels. Free chondroitin sulfate was obtained by papain digestion of knee joint cartilage of 10 week old pigs by the method of Simunek and Muir (7). Heparan sulfate, which contained approximately one sulfate group per tetrasaccharide repeating unit, was isolated from human aorta (19). Hyaluronic acid (potassium salt, Koch Light Laboratories Ltd., Colnbrook, England) was obtained from human umbilical cord and the keratan sulfate from bovine invertebral disc. Populations of proteoglycans of different molecular size were isolated from knee joint cartilage of 10 week old sound and lame pigs, by extraction first. with 0.15&! sodium acetate (20) and then with 2 M CaCl, (21) by the method of Simunek and Muir (7). Digestion of proteoglycans with trypsin releases small peptide fragments to which are attached two glycosaminoglycan chains (22). Laryngeal cartilage (6.852 gm, wet weight) was incubated with 0.5 mg of trypsin (British Drug Houses, 3000 NF units/mg) in 25 ml of 0.04 citrate-phosphate, pH 7.9, for 16 hr at 37°C (10). The mixture was then clarified by centrifugation at 2130 g for 10 min and the chondroitin sulfate “doublet,s” were precipitated with 9-aminoacridine and subsequently isolated as t‘heir sodium salts (23). Hascall and Sajdera (24) reported that bovine nasal septum proteoglycans may be disaggregated in the presence of 4 M guanidine hydrochloride into a protein rich and uranic acid rich fraction. A proteoglycan extract, comparable to that fraction described by Hascall and Sajdera as “aggregate,” was prepared from 9 month old pig knee joint cartilage by extraction for 48 hr in 4 M gunnidine hydrochloride, dialysis against 9 vol of water to bring the guanidine concentration to 0.5 M and equilibrium density centrifugation (98,000g) in cesium chloride (starting density, p = 1.5 gm/ml) for 48 hr. The “aggregate” was isolated from the bottom two-fifths of the density gradient. The disaggregated proteoglycan fraction was prepared by equilibrium centrifugation of the ‘laggregatr” in a similar density gradient (starting density, p = 1.5 gm/ml) containing 4N guanidine hydrochloride and isolat,ed from the bottom t.wo-seventeent.hs of the gradient.
616
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MUIR
RESULTS
Preliminary trials with gels containing 0.5% agarose and 0.5% acrylamide demonstrated that the strength of the gels varied somewhat with different batches of agarose as supplied by British Drug Houses. The strength of these gels was considerably improved and reproducibility attained by increasing the agarose content to 0.6%. The mobilities of single chondroitin sulfate chains obtained by papain digestion of proteoglycans were identical on gels comprised of 0.6% agarose with the content of polyacrylamide varying from 0.6 to 0.8%. Increasing this to more than O.S%, however, resulted in progressive decreases in the mobility of the chondroitin sulfate. The concentration of polyacrylamide chains below a total monomer concentration of 0.8% was thus too low to produce significant fractional resistance to the migration of single chondroitin sulfate chains. The molecular sieving properties of the composite gel were investigated by comparing the mobilities of free chondroitin sulfate (papain digest), the chondroitin sulfate “doublet” (trypsin digest) (22), the sodium acetate extract, and the calcium chloride extract of articular cartilage. These four preparations represent molecular populations with
Summary
of Gel
Chromatographic Glycosaminoglycans
TABLE 1 and Gel Electrophoretic and Proteoglycans
Properties
Gel chromatography ‘$& retarded
Samples
from
pig cartilage
Glycosaminoglycans: Chondrotin sulfate Chondroitin sulfate “doublet” Proteoglycans from knee joint cartilage: Sodium acetate ext. (normal pigs)” Calcium chloride ext. (normal pigs) Calcium chloride ext. (lame pigs)b
Sephadex G-200
100 0 0 0 0
6% agarose
of
Polyacrylamideagarose electrophoresis Sepha rose 4B Relative
-
-
47 32 77
81 70 95
mobility
100.0 89.7 74.0 0, 37.0, 41.7c 81.0
The electrophoresis was performed in a polyacrylamide (1.2a/,)-agarose with a 0.04 M T&acetate buffer, pH 6.8. Relative mobility = distance migrated by band expressed as percentage traveled by chondroitin sulfate. a Gel chromatographic properties t,aken from Simunek and Muir (7). b From Simunek and Muir (8). c Three bands.
(0.6y0)
gel
of distance
GEL
ELECTROPHORESIS
OF
PROTEOGLYCANS
617
similar charge densities but considerably different hydrodynamic sizes as assessed by gel chromatography (Table 1). Chondroitin sulfate was retarded and the “doublet” excluded on Sephadex G-200. Sequential extraction of articular cartilage brings into solution proteoglycans of increasing size, the smaller being extracted with sodium acetate, 47% of which are retarded on Sepharose 6B (7). The larger proteoglycans are extracted with 2M CaCl,, some of which are large enough to be excluded from Sepharose 2B (7).
B
C
D
E
FIG. 1. Densitometric traces of glycosaminoglycans and proteoglycans of different hydrodynamic size: (A) Chondroitin sulfate (papain digest). (B) Chondroitin sulfate “doublet” (trypsin digest). (C) 0.15 M sodium acetate preparation of 10 week old pig knee joint cartilage. (D) 2M CaCL proteoglycan preparation of pig knee joint cartilage. (E) 2M CaCL proteoglycan preparation from knee joint cartilage of lame pigs. Gel composition : polyacrylamide 1.2%, agarose 0.6%. Buffer: 0.04 M Tris-acetate, pH 6.8.
618
MCDEVITT
AND
MUIR
The gel concentration that was most satisfactory in producing differences in mobility according to hydrodynamic size of these four preparations consisted of a mixture of 0.6% agarose with 1.2% acrylamide. Figure 1 shows that the mobility of single chondroitin sulfate chains (A) was greater than the double chains (B), while the mobilities of proteoglycans decreased with size. The CaCl, extract of the 10 week old pig femoral condylar cartilage contained some molecules too large to penetrate the gel and gave a deeply stained band at the top of the gel. The remainder, which did penetrate the gel (D), migrated more slowly than those molecules extracted with sodium acetate (C). In contrast, Fig. 1 shows that the proteoglycans in corresponding calcium chloride extracts of cartilage of lame pigs were all sufficiently small to penetrate the gel and had higher mobilities (E) than those that did
and disaggregated proteoglycans isoFIG. 2. Gel electrophoresis of “aggregates” lated by equilibrium density centrifugation in cesium chloride gradients: (A) Chondroitin sulfate. (B) Disaggregated proteoglycan. (C) Starting material containing acrylamide 0.8%. Buffer: 0.04 M “aggregates.” Gel composition : agarose 0.6%, Tris-acetate, pH 6.8.
GEL
ELECTROPHORESIS
OF
PROTEOGLTCANS
619
so from the cartilage of sound pigs (U) that were litter mates of the same age. The proteoglycans from the lame group of pigs were shown to be of smaller hydrodynamic size than those from the sound group when compared on gel chromatography (8). The gel electrophoretic behavior obtained with the ‘(aggregate” and dissociated proteoglycans are illustrated in Fig. 2. All the disaggregated fraction penetrated the gel. The “aggregate” fraction, however, separated into two components, a fraction too large in hydrodynamic size to penetrate the gel and a component with mobility similar to the disaggregat.ed extract. A preliminary study of the gel electrophoresis of glycosaminoglycans demonstrated that chondroitin sulfate keratan sulfate, heparan sulfate, and hyaluronic acid had different migration rates on gels comprised of 0.6% agarose and 1.5% acrylamide (Fig. 3). Chondroitin sulfate displayed faster mobility than keratan sulfate and heparan sulfate whose charge densities were lower. Keratan sulfate is considered to contain
FIG. 3. Electrophoresis of glycosaminoglycans on polyacrylamide (1.5%)~agarose (0.6%) gels in 0.02M citrate-phosphate, pH 3.15, buffer: (A) Chondroitin sulfate. (B) Chondroitin sulfate “doublet” trypsin digest). CC) Iieratin sulfate. (D) Human aorta heparan sulfate. (E) Mixture of glycosaminoglycans from hippopotamus aorta. (F) Hyaluronic acid.
620
MCDEVITT
AND
MUIR
no uranic acid, while the purified heparan sulfate from human aorta possessed only about one sulfate group per tetrasaccharide (19). The glycosaminoglycans from hippopotamus aorta separated into three bands, one of which was similar in migration to the more purified single band of human aorta heparan sulfate. Band sharpening was considerably improved, except in the case of the keratan sulfate, by lowering the pH of the gel and electrode buffers to 3.15. The carboxyl residues of the uranic acids are approximately half titrated at this pH. The diffuse band obtained with the keratan sulfate fraction suggested a high degree of polydispersity in size and/or sulfate content in these molecules (25). The extremely low mobility of hyaluronic acid in these gels at pH 3.5 is consistent with the large hydrodynamic size and absence of sulfate residues in this compound (26). DISCUSSION
Hendrick and Smith (14) have demonstrated that the mobility of a protein on polyacrylamide gels is a function of both size and charge of the molecule. Composite agarose (0.5%)-acrylamide (3.5%) gels have been shown to function as molecular sieves in the separation of high molecular weight RNA mixtures (15). The currently proposed model of proteoglycan macromoleculas structure consists of a protein core to which are attached glycosaminoglycan chains of chondroitin sulfate and keratan sulfate (reviewed in 27). Although the keratan sulfate content of articular cartilage proteoglycans is variable (6), their charge density is predominantly a property of chondroitin sulfate, their major constituent. The composite gel electrophoretic mobilities of chondroitin sulfate chains and proteoglycan extracts were inversely related to their respective hydrodynamic sizes as assessed by gel chromatography (Table 1). The polyacrylamide-agarose gels therefore possess the property of being able to function as molecular sieves in the electrophoresis of proteoglycans. At acrylamide concentrations of 1.2% (0.6% agarose), the gels could further distinguish between single chondroitin sulfate chains (papain digest) having 20-26 repeating disaccharide units (6) and double chains (trypsin digest), where each chain had a similar average number of repeating disaccharide units (28). The quality of the band sharpening obtained with different glycosaminoglycan extracts was superior to the published separations of Hilborn and Anastassiadis (11). The diffuse bands they obtained were attributed to the sensitivity of the relatively small pore gel (6% acrylamide) to size polydispersity in the glycosaminoglycan chains (29). A considerable advantage of the present system is that the porosity of the gels and the pH of the buffer may be varied to suit the requirements
GEL
ELECTROPHORESIS
OF
PROTEOGLYCANS
621
of a particular investigation. Appropriate control of these two parameters could provide separation of any two glycosaminoglycans which differ in size or charge density. The need for new micro methods sensitive enough for the analysis of glycosaminogIycans from welI-defined histological structures has been stressed, as these compounds show considerable topographic variation at tissue level (30). Variation with depth from the articular surface of human femoral condyles in the contents of chondroitin sulfate and keratan sulfate has been noted (31). Since the amount of proteoglycan required for each electrophoresis tube is only 2-20 pg, possible topographic variation in their size could be assessed by this method. Furthermore, the method may be used to detect aggregates of proteoglycans, the presence of which is otherwise demonstrable only by analyt,ical ultracentrifugation (24). Reproducible mobilities were obtained on the composite polyacrylamide-agarose gel, even with different batches of agarose as supplied by British Drug Houses. The use of Perspex tubes and stringent adherence to the conditions described in the “gel preparation” and “electrophoretic conditions” sections are, however, very important for reproducibility. SUMMARY
The electrophoresis of proteoglycans and glycosaminoglycans on largepore polyacrylamide gels which contained agarose as a support medium is described. The average pore diameter of the gels was sufficient to allow penetration of all but the very largest proteoglycans of articular cartilage, making it possible to distinguish “aggregates” from disaggregated molecules. The relative sizes of proteoglycans could be assessed, using only microgram quantities on these gels, as the mobilities were inversely related to hydrodynamic size as determined by gel chromatography. The method was able to separate single from double chains of chondroitin sulfate. On the other hand, the relative mobilities of separate chains of different types of glycosaminoglycans were a function of their charge density. The method has been applied to a pathological condition in which changes in the sizes of proteoglycans were evident. ACKNOWI,EDGMEN1‘R Samples of proteoglycans from sound and lame pig articular cartilage werr kindly supplied by Dr. Z. Simunek. Protcoglycan- b isolated by equilibrium eentrifugntion in cesium chloride density gradients were n gift froln Dr. C. P. Tsiganos. Onr of 11s (C.M.) acknowledges financial support from the Agricultural Research Counc~il and we thank the Arthritis and Rhrumntism Council for general support.
622
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REFERENCES 1. MUIR, H., in “The Chemical Physiology of Mucopolysaccharides” (G. Quintarelli, ed.), p. 1. Little, Brown, Boston, 1967. 2. FRANEK, M. D., AND DUNSTONE, J., J. Biol. Chem. 242, 3460 (1967). 3. HOFFMAN, P., MASHBURN, T. A., MEYER, K., AND BRAY, B. A., J. Bill. 242, 3799 (1967). 4. TSIQANOS, C. P., AND Mum, H., in “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.). Vol. 2, p. 859. Academic Press, London ( 1970) . 5. TSIGANOS, C. P., HARDINGHAM, T. E., AND Mum, H., Biochim. Biophys. Acta 229, 529 (1971). 6. BRANDT, K. D., AND Mum, H., Biochem. J. 114, 871 (1969). 7. SIMUNEK, Z., AND Mum, H., Biochem. J., in press. 8. SIMUNEK, Z., AND Mum, H., Abstr. VIZ Buro. Rheumatol. Congr., Brighton (1972) and Bioehem. J., submitted. 9. MATHEWS, M. B., AND LOZAIT’ITE, I., Arch. Biochem. Biophys. 74, 158 (1958). 10. LUSCOMBE, M., AND PHELPS, C. F., Biochem. J. 103, 103 (1967). 11. HILBORN, J. C., AND ANASTAGSIADIS, P. A., Anal. B&hem. 31, 51 (1969). 12. BARRETT, A. J., Nature 211, 1188 (1966). 13. ORNSTFIN, L., Ann. N. Y. Acad. Sci. 121, 321 (1964). 14. HENDRICK, J. L., AND SMITH, A. J., Arch. Biochem. Biophys. 126, 155 (1968). 15. PEACOCK, A. C., AND DINQMAN, C. W., Biochemistry 7, 668 (1968). 16. LQENINU, U. E., B&hem. J. 10% 251 (1967). 17. TOMBS, M. P., in “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.), Vol. 2, p. 443. Heinemann, London, 1968; Wiley, New York. 18. SCOTT, J. E., AND DORLING, J., Histochemie 5, 221 (1965). 19. Mum, II., in ‘Structure and Function of Connective and Skeletal Tissue” (S. FittonJackson, R. D. Harkness, S. M. Partridge, and G. V. Trustram, eds.), p. 137. Butterworth, London, 1965. 20. TSIQANOS, C. P., AND MXIIR, H., Biochem. J. 113, 879 (1969). 21. SAJDERA, S. W., AND HASCALL, V. C., J. Biol. Chem. 244, 77 (1969). 22. MATHEWS, M. B., Fed. Proc. 27, 529 (1968). 23. Mum, H., Biochem. J. 69, 195 (1958). 24. HASCALL, V. C., AND SAJDERA, S. W., J. Biol. Chem. 244, 2384 (1969). 25. MATHEWS, M. B., AND GLAGOV, S., J. Clin. Invest. 45, 1103 (1966). 26. OGSTON, A. G., AND STANIER, J. E., Biochem. J. 49, 585 (1951). 27. Mum, H., Amer. J. Med. 47, 673 (1969). 28. TSIGANOS, C. P., AND MUIR, H., Biochem. J. 113, 885 (1969). 29. HILBORN, J. C., AND ANASTASSIADIS, P. A., Anal. Biochem. 39, 88 (1971). 30. SZIRMAI, J. A., VAN BOVEN-DE TYSSONSK, E., AND GARDELL, S., B&him. Biophys. Acta 136, 331 (1967). 31. MAROUDAS, A., MUIR, H., AND WINGHAM, J., Biochim. Biophys. Acta 177, 492 (1969).
BIOCHEMISTRY
Gel
44,
Electrophoresis aminoglycans
612-622 (1971)
of Proteoglycans on
Large-Pore
Polyacrylamide-Agarose C. A. McDEVITT Kennedy
Institute
AND HELEN
and
Glycos-
Composite Gels MUIR
of Rheumatology, Bute Gardens, Hammersmith, London, W. 6, United Kingdom
Received May 5, 1971; revised July 2, 1971
The heterogeneity of proteoglycans of cartilage is now well established (l-5). Articular cartilage contains populations of proteoglycans of different size, the relative proportions of which vary with the age and pathological state of the tissue (6-8). The large Stokes radii (9,lO) of these hydrated molecules have made it difficult to apply sensitive highresolution separation techniques in the study of their heterogeneity. Although glycosaminoglycans (“acidic mucopolysaccharides”) have been separated in 6% polyacrylamide gels (11)) the intact proteoglycans are completely immobilized in 7% gels (12). Polyacrylamide gel electrophoresis has not been applied before to proteoglycans. Pore size is inversely related to the concentration of acrylamide (13) but polyacrylamide does not form gels below concentrations of 2% (14). Peacock and Dingman (15) have, however, succeeded in forming 1.5% polyacrylamide gels to separate high molecular weight RNA mixtures by incorporating 0.5% agarose into the gel medium for support. By modifying the technique of Peacock and Dingman, we have been able to form stable gels, comprised of 0.5-1.50/o acrylamide and 0.6% agarose, which are sufficiently porous to enable all but the very largest proteoglycans to penetrate them. Glycosaminoglycans migrate on these gels as sharp bands, the mobilities of which are a function of their charge density, whereas proteoglycans which are of similar charge density display mobilities that are inversely related to their molecular size. METHODS
Gel Preparation Acrylamide and N,N’-methylenebisacrylamide were recrystallized from chloroform and acetone, respectively, by the method of Loening 612
GEL
ELECTROPHORESIS
OF
PROTEOGLYCANS
613
(16). The buffer used was 0.04 1M Tris-acetate containing 1 mM sodium sulfate (final concentrations) adjusted at 4°C to pH 6.8 with acetic acid. Tris-acetate was used instead of the chloride to avoid formation of hypochlorite by electrolysis (16). The presence of sodium sulfate in the buffer prevented adsorption of the glyeosaminoglycans on to the composite gel matrix which would otherwise occur. The gels consisted in the main of 1.2% acrylamide and 0.6% agarose and were prepared as follows: Agarose, 0.48 gm (British Drug Houses, Ltd, Poole, Dorset, England, ‘
614
MCDEVITT
AND
MUIR
gels were then carefully replaced in the Perspex tubes, which were resealed at one end with Visking tubing and Parafilm. Electrophoretic
Conditions
The electrode buffer consisted of the Tris buffer diluted four times, to which EDTA was added to a concentration of 1 m.M to eliminate the possibility of heavy met,als affecting the electrophoresis of the proteoglycans (16). When electrophoresis was performed at low pH, the electrode buffer was a 1: 10 dilution of the 0.02 M citrate phosphate buffer and EDTA was again added to a concentration of 1 mM. The volume of the anode buffer was 400 ml and the volume of the cathode buffer 250 ml. The Perspex tubes with the Parafilm removed were placed vertically in the electrophoresis chamber (Pleuger S. A., Winjnegem, Belgium), and to ensure adequate cooling they were immersed up to the top of the gels in the buffer of the anode compartment. A current of 4 mA/tube (200 V) was applied for 1 hr to remove unpolymerized acrylamide and catalysts from the gels. Fresh buffer was then poured into the electrode compartments and samples of proteoglycans (2-20 pg) in 20 all of a 40% sucrose-water solution were layered on to the top of the gels. A voltage gradient of 13.8 V/cm was applied until a bromophenol blue marker entered the gel. The current was then increased to 4 mA/tube for the duration of the run (about 60 min) . The voltage gradient was about 27.8 V/cm. The temperature of both electrode buffers was 7.6”C before electrophoresis. The temperature of the cathode buffer rose to 11.5” and that of the anode buffer remained constant after 1 hr of electrophoresis. It was essential that electrophoresis was performed in the cold room and that the current did not rise above 5 mA/tube. Localization
of Bands
The gels were removed from their Perspex tubes and stained for 15 min in 0.2% toluidine blue in 0.1 N acetic acid. This dye forms waterinsoluble metachromatic complexes with proteoglycans (18). The excess dye was removed by immersing the gels in 3% acetic acid for 90 min, followed by 34 repeated washes in water and then overnight in water. The gels were then scanned with a Locarte gel densitometer using a filter allowing maximum transmission at 520 nm (Ilford filter No. 624). Neither Alcian blue nor acridine orange was satisfactory because they stained the composite gel matrix too strongly and were not removed during destaining. The distance from the base of the meniscus at the top of the gel to the point of maximum intensity of each band was measured in millimeters from the densitometric scans. A sample of articular cartilage chondroitin sulfate was included in all
GEL ELECTROPHORESIS
OF PROTEOGLYCAhTS
runs as a reference standard. The relative mobility expressed as a percentage of the distance migrated fate under the same conditions.
615
of each band was by chondroitin sul-
Source of Proteoglycans and Glycosaminoglycans
A comparative study of the mobilities of chondroitin sulfate, heparan sulfate, and hyaluronic acid was undertaken to assess the affect of charge density on the mobilities of glycosaminoglycans on the composite gels. Free chondroitin sulfate was obtained by papain digestion of knee joint cartilage of 10 week old pigs by the method of Simunek and Muir (7). Heparan sulfate, which contained approximately one sulfate group per tetrasaccharide repeating unit, was isolated from human aorta (19). Hyaluronic acid (potassium salt, Koch Light Laboratories Ltd., Colnbrook, England) was obtained from human umbilical cord and the keratan sulfate from bovine invertebral disc. Populations of proteoglycans of different molecular size were isolated from knee joint cartilage of 10 week old sound and lame pigs, by extraction first. with 0.15&! sodium acetate (20) and then with 2 M CaCl, (21) by the method of Simunek and Muir (7). Digestion of proteoglycans with trypsin releases small peptide fragments to which are attached two glycosaminoglycan chains (22). Laryngeal cartilage (6.852 gm, wet weight) was incubated with 0.5 mg of trypsin (British Drug Houses, 3000 NF units/mg) in 25 ml of 0.04 citrate-phosphate, pH 7.9, for 16 hr at 37°C (10). The mixture was then clarified by centrifugation at 2130 g for 10 min and the chondroitin sulfate “doublet,s” were precipitated with 9-aminoacridine and subsequently isolated as t‘heir sodium salts (23). Hascall and Sajdera (24) reported that bovine nasal septum proteoglycans may be disaggregated in the presence of 4 M guanidine hydrochloride into a protein rich and uranic acid rich fraction. A proteoglycan extract, comparable to that fraction described by Hascall and Sajdera as “aggregate,” was prepared from 9 month old pig knee joint cartilage by extraction for 48 hr in 4 M gunnidine hydrochloride, dialysis against 9 vol of water to bring the guanidine concentration to 0.5 M and equilibrium density centrifugation (98,000g) in cesium chloride (starting density, p = 1.5 gm/ml) for 48 hr. The “aggregate” was isolated from the bottom two-fifths of the density gradient. The disaggregated proteoglycan fraction was prepared by equilibrium centrifugation of the ‘laggregatr” in a similar density gradient (starting density, p = 1.5 gm/ml) containing 4N guanidine hydrochloride and isolat,ed from the bottom t.wo-seventeent.hs of the gradient.
616
MCDEVITT
AND
MUIR
RESULTS
Preliminary trials with gels containing 0.5% agarose and 0.5% acrylamide demonstrated that the strength of the gels varied somewhat with different batches of agarose as supplied by British Drug Houses. The strength of these gels was considerably improved and reproducibility attained by increasing the agarose content to 0.6%. The mobilities of single chondroitin sulfate chains obtained by papain digestion of proteoglycans were identical on gels comprised of 0.6% agarose with the content of polyacrylamide varying from 0.6 to 0.8%. Increasing this to more than O.S%, however, resulted in progressive decreases in the mobility of the chondroitin sulfate. The concentration of polyacrylamide chains below a total monomer concentration of 0.8% was thus too low to produce significant fractional resistance to the migration of single chondroitin sulfate chains. The molecular sieving properties of the composite gel were investigated by comparing the mobilities of free chondroitin sulfate (papain digest), the chondroitin sulfate “doublet” (trypsin digest) (22), the sodium acetate extract, and the calcium chloride extract of articular cartilage. These four preparations represent molecular populations with
Summary
of Gel
Chromatographic Glycosaminoglycans
TABLE 1 and Gel Electrophoretic and Proteoglycans
Properties
Gel chromatography ‘$& retarded
Samples
from
pig cartilage
Glycosaminoglycans: Chondrotin sulfate Chondroitin sulfate “doublet” Proteoglycans from knee joint cartilage: Sodium acetate ext. (normal pigs)” Calcium chloride ext. (normal pigs) Calcium chloride ext. (lame pigs)b
Sephadex G-200
100 0 0 0 0
6% agarose
of
Polyacrylamideagarose electrophoresis Sepha rose 4B Relative
-
-
47 32 77
81 70 95
mobility
100.0 89.7 74.0 0, 37.0, 41.7c 81.0
The electrophoresis was performed in a polyacrylamide (1.2a/,)-agarose with a 0.04 M T&acetate buffer, pH 6.8. Relative mobility = distance migrated by band expressed as percentage traveled by chondroitin sulfate. a Gel chromatographic properties t,aken from Simunek and Muir (7). b From Simunek and Muir (8). c Three bands.
(0.6y0)
gel
of distance
GEL
ELECTROPHORESIS
OF
PROTEOGLYCANS
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similar charge densities but considerably different hydrodynamic sizes as assessed by gel chromatography (Table 1). Chondroitin sulfate was retarded and the “doublet” excluded on Sephadex G-200. Sequential extraction of articular cartilage brings into solution proteoglycans of increasing size, the smaller being extracted with sodium acetate, 47% of which are retarded on Sepharose 6B (7). The larger proteoglycans are extracted with 2M CaCl,, some of which are large enough to be excluded from Sepharose 2B (7).
B
C
D
E
FIG. 1. Densitometric traces of glycosaminoglycans and proteoglycans of different hydrodynamic size: (A) Chondroitin sulfate (papain digest). (B) Chondroitin sulfate “doublet” (trypsin digest). (C) 0.15 M sodium acetate preparation of 10 week old pig knee joint cartilage. (D) 2M CaCL proteoglycan preparation of pig knee joint cartilage. (E) 2M CaCL proteoglycan preparation from knee joint cartilage of lame pigs. Gel composition : polyacrylamide 1.2%, agarose 0.6%. Buffer: 0.04 M Tris-acetate, pH 6.8.
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The gel concentration that was most satisfactory in producing differences in mobility according to hydrodynamic size of these four preparations consisted of a mixture of 0.6% agarose with 1.2% acrylamide. Figure 1 shows that the mobility of single chondroitin sulfate chains (A) was greater than the double chains (B), while the mobilities of proteoglycans decreased with size. The CaCl, extract of the 10 week old pig femoral condylar cartilage contained some molecules too large to penetrate the gel and gave a deeply stained band at the top of the gel. The remainder, which did penetrate the gel (D), migrated more slowly than those molecules extracted with sodium acetate (C). In contrast, Fig. 1 shows that the proteoglycans in corresponding calcium chloride extracts of cartilage of lame pigs were all sufficiently small to penetrate the gel and had higher mobilities (E) than those that did
and disaggregated proteoglycans isoFIG. 2. Gel electrophoresis of “aggregates” lated by equilibrium density centrifugation in cesium chloride gradients: (A) Chondroitin sulfate. (B) Disaggregated proteoglycan. (C) Starting material containing acrylamide 0.8%. Buffer: 0.04 M “aggregates.” Gel composition : agarose 0.6%, Tris-acetate, pH 6.8.
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so from the cartilage of sound pigs (U) that were litter mates of the same age. The proteoglycans from the lame group of pigs were shown to be of smaller hydrodynamic size than those from the sound group when compared on gel chromatography (8). The gel electrophoretic behavior obtained with the ‘(aggregate” and dissociated proteoglycans are illustrated in Fig. 2. All the disaggregated fraction penetrated the gel. The “aggregate” fraction, however, separated into two components, a fraction too large in hydrodynamic size to penetrate the gel and a component with mobility similar to the disaggregat.ed extract. A preliminary study of the gel electrophoresis of glycosaminoglycans demonstrated that chondroitin sulfate keratan sulfate, heparan sulfate, and hyaluronic acid had different migration rates on gels comprised of 0.6% agarose and 1.5% acrylamide (Fig. 3). Chondroitin sulfate displayed faster mobility than keratan sulfate and heparan sulfate whose charge densities were lower. Keratan sulfate is considered to contain
FIG. 3. Electrophoresis of glycosaminoglycans on polyacrylamide (1.5%)~agarose (0.6%) gels in 0.02M citrate-phosphate, pH 3.15, buffer: (A) Chondroitin sulfate. (B) Chondroitin sulfate “doublet” trypsin digest). CC) Iieratin sulfate. (D) Human aorta heparan sulfate. (E) Mixture of glycosaminoglycans from hippopotamus aorta. (F) Hyaluronic acid.
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no uranic acid, while the purified heparan sulfate from human aorta possessed only about one sulfate group per tetrasaccharide (19). The glycosaminoglycans from hippopotamus aorta separated into three bands, one of which was similar in migration to the more purified single band of human aorta heparan sulfate. Band sharpening was considerably improved, except in the case of the keratan sulfate, by lowering the pH of the gel and electrode buffers to 3.15. The carboxyl residues of the uranic acids are approximately half titrated at this pH. The diffuse band obtained with the keratan sulfate fraction suggested a high degree of polydispersity in size and/or sulfate content in these molecules (25). The extremely low mobility of hyaluronic acid in these gels at pH 3.5 is consistent with the large hydrodynamic size and absence of sulfate residues in this compound (26). DISCUSSION
Hendrick and Smith (14) have demonstrated that the mobility of a protein on polyacrylamide gels is a function of both size and charge of the molecule. Composite agarose (0.5%)-acrylamide (3.5%) gels have been shown to function as molecular sieves in the separation of high molecular weight RNA mixtures (15). The currently proposed model of proteoglycan macromoleculas structure consists of a protein core to which are attached glycosaminoglycan chains of chondroitin sulfate and keratan sulfate (reviewed in 27). Although the keratan sulfate content of articular cartilage proteoglycans is variable (6), their charge density is predominantly a property of chondroitin sulfate, their major constituent. The composite gel electrophoretic mobilities of chondroitin sulfate chains and proteoglycan extracts were inversely related to their respective hydrodynamic sizes as assessed by gel chromatography (Table 1). The polyacrylamide-agarose gels therefore possess the property of being able to function as molecular sieves in the electrophoresis of proteoglycans. At acrylamide concentrations of 1.2% (0.6% agarose), the gels could further distinguish between single chondroitin sulfate chains (papain digest) having 20-26 repeating disaccharide units (6) and double chains (trypsin digest), where each chain had a similar average number of repeating disaccharide units (28). The quality of the band sharpening obtained with different glycosaminoglycan extracts was superior to the published separations of Hilborn and Anastassiadis (11). The diffuse bands they obtained were attributed to the sensitivity of the relatively small pore gel (6% acrylamide) to size polydispersity in the glycosaminoglycan chains (29). A considerable advantage of the present system is that the porosity of the gels and the pH of the buffer may be varied to suit the requirements
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of a particular investigation. Appropriate control of these two parameters could provide separation of any two glycosaminoglycans which differ in size or charge density. The need for new micro methods sensitive enough for the analysis of glycosaminogIycans from welI-defined histological structures has been stressed, as these compounds show considerable topographic variation at tissue level (30). Variation with depth from the articular surface of human femoral condyles in the contents of chondroitin sulfate and keratan sulfate has been noted (31). Since the amount of proteoglycan required for each electrophoresis tube is only 2-20 pg, possible topographic variation in their size could be assessed by this method. Furthermore, the method may be used to detect aggregates of proteoglycans, the presence of which is otherwise demonstrable only by analyt,ical ultracentrifugation (24). Reproducible mobilities were obtained on the composite polyacrylamide-agarose gel, even with different batches of agarose as supplied by British Drug Houses. The use of Perspex tubes and stringent adherence to the conditions described in the “gel preparation” and “electrophoretic conditions” sections are, however, very important for reproducibility. SUMMARY
The electrophoresis of proteoglycans and glycosaminoglycans on largepore polyacrylamide gels which contained agarose as a support medium is described. The average pore diameter of the gels was sufficient to allow penetration of all but the very largest proteoglycans of articular cartilage, making it possible to distinguish “aggregates” from disaggregated molecules. The relative sizes of proteoglycans could be assessed, using only microgram quantities on these gels, as the mobilities were inversely related to hydrodynamic size as determined by gel chromatography. The method was able to separate single from double chains of chondroitin sulfate. On the other hand, the relative mobilities of separate chains of different types of glycosaminoglycans were a function of their charge density. The method has been applied to a pathological condition in which changes in the sizes of proteoglycans were evident. ACKNOWI,EDGMEN1‘R Samples of proteoglycans from sound and lame pig articular cartilage werr kindly supplied by Dr. Z. Simunek. Protcoglycan- b isolated by equilibrium eentrifugntion in cesium chloride density gradients were n gift froln Dr. C. P. Tsiganos. Onr of 11s (C.M.) acknowledges financial support from the Agricultural Research Counc~il and we thank the Arthritis and Rhrumntism Council for general support.
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REFERENCES 1. MUIR, H., in “The Chemical Physiology of Mucopolysaccharides” (G. Quintarelli, ed.), p. 1. Little, Brown, Boston, 1967. 2. FRANEK, M. D., AND DUNSTONE, J., J. Biol. Chem. 242, 3460 (1967). 3. HOFFMAN, P., MASHBURN, T. A., MEYER, K., AND BRAY, B. A., J. Bill. 242, 3799 (1967). 4. TSIQANOS, C. P., AND Mum, H., in “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.). Vol. 2, p. 859. Academic Press, London ( 1970) . 5. TSIGANOS, C. P., HARDINGHAM, T. E., AND Mum, H., Biochim. Biophys. Acta 229, 529 (1971). 6. BRANDT, K. D., AND Mum, H., Biochem. J. 114, 871 (1969). 7. SIMUNEK, Z., AND Mum, H., Biochem. J., in press. 8. SIMUNEK, Z., AND Mum, H., Abstr. VIZ Buro. Rheumatol. Congr., Brighton (1972) and Bioehem. J., submitted. 9. MATHEWS, M. B., AND LOZAIT’ITE, I., Arch. Biochem. Biophys. 74, 158 (1958). 10. LUSCOMBE, M., AND PHELPS, C. F., Biochem. J. 103, 103 (1967). 11. HILBORN, J. C., AND ANASTAGSIADIS, P. A., Anal. B&hem. 31, 51 (1969). 12. BARRETT, A. J., Nature 211, 1188 (1966). 13. ORNSTFIN, L., Ann. N. Y. Acad. Sci. 121, 321 (1964). 14. HENDRICK, J. L., AND SMITH, A. J., Arch. Biochem. Biophys. 126, 155 (1968). 15. PEACOCK, A. C., AND DINQMAN, C. W., Biochemistry 7, 668 (1968). 16. LQENINU, U. E., B&hem. J. 10% 251 (1967). 17. TOMBS, M. P., in “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.), Vol. 2, p. 443. Heinemann, London, 1968; Wiley, New York. 18. SCOTT, J. E., AND DORLING, J., Histochemie 5, 221 (1965). 19. Mum, II., in ‘Structure and Function of Connective and Skeletal Tissue” (S. FittonJackson, R. D. Harkness, S. M. Partridge, and G. V. Trustram, eds.), p. 137. Butterworth, London, 1965. 20. TSIQANOS, C. P., AND MXIIR, H., Biochem. J. 113, 879 (1969). 21. SAJDERA, S. W., AND HASCALL, V. C., J. Biol. Chem. 244, 77 (1969). 22. MATHEWS, M. B., Fed. Proc. 27, 529 (1968). 23. Mum, H., Biochem. J. 69, 195 (1958). 24. HASCALL, V. C., AND SAJDERA, S. W., J. Biol. Chem. 244, 2384 (1969). 25. MATHEWS, M. B., AND GLAGOV, S., J. Clin. Invest. 45, 1103 (1966). 26. OGSTON, A. G., AND STANIER, J. E., Biochem. J. 49, 585 (1951). 27. Mum, H., Amer. J. Med. 47, 673 (1969). 28. TSIGANOS, C. P., AND MUIR, H., Biochem. J. 113, 885 (1969). 29. HILBORN, J. C., AND ANASTASSIADIS, P. A., Anal. Biochem. 39, 88 (1971). 30. SZIRMAI, J. A., VAN BOVEN-DE TYSSONSK, E., AND GARDELL, S., B&him. Biophys. Acta 136, 331 (1967). 31. MAROUDAS, A., MUIR, H., AND WINGHAM, J., Biochim. Biophys. Acta 177, 492 (1969).