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The determination of small quantities of isomeric chondroitin sulfates
BIOCHIMICA ET BIOPHYSICA ACTA
137
Preliminary Notes PN 81000
The determination of small quantities of isomeric chondroitin sulfates A new separation method for acid mucopolysaccharides was reported in a previous paper from this laboratory 1. Although a separation of extremely small amounts of connective-tissue mucopolysaccharides is possible by this method, the isomeric chondroitin sulfates remain unresolved. This deficiency also characterizes other presently available separation methods 2-5, although a partial separation of individual isomers m a y be obtained. Dermatan sulfate and chondroitin sulfate B are easily separated from the other two isomers b y electrophoresis at low pHe. Chondroitin 4-sulfate (chondroitin sulfate A) and dermatan sulfate (D) m a y be separated from one another by exploiting the different solubility of their Zn salts v. Chondroitin 4-sulfate and chondroitin 6-sulfate (chondroitin sulfate C) differ so little in their physical and chemical properties that a satisfactory separation of these isomers is difficults, or impossible. Lately MATHEWS9 described an elegant method for the determination of chondroitin sulfates of Type C, based on the observation that N-acetylhexosamines substituted only in position 6 behave normally in the Morgan-Elson reaction, while a substitution in position 4 causes an almost complete suppression of the color formation 1°-1~. The "acetylhexosamines" are released from chondroitin 4-sulfate and chondroitin 6-sulfate by the action of testicular hyaluronidase. This paper describes a procedure for the simultaneous determination of the isomeric chondroitin sulfates isolated from various biological materials. This procedure is founded on the following observations. (I) The known selective action of testicular hyaluronidase on chondroitin sulfates. Under the influence of this enzyme only the chondroitin-4-sulfate and chondroitin-6-sulfate isomers are split, leaving dermatan sulfate unaffected s, 14,15. (2) The different distribution coefficients, Ka, for the undigested dermatan sulfate molecule and the "tetrasaccharides" which are the main end products of chondroitin4-sulfate and chondroitin-6-sulfate degradation. (3) The anomalous behavior of Nacetylgalactosamine 4-sulfate in the Morgan-Elson reaction as mentioned above 9. I.O ml of the mixture containing only the isomeric chondroitin sulfates (50-500/~g of each isomer as ghicuronic acid) was mixed with an equal volume of citrate-phosphate buffer as described 9. I.O ml of a solution of testicular hyaluronidase in o.I M NaC1 containing lOO-6OO I.U. of the enzyme (Kinetin, Schering AG, Berlin, Germany) was added. This mixture was incubated over a 24-h period in glass-stoppered tubes at 38-4 o°. Although this reaction can be completed in a shorter time 9, a longer period was necessary because dermatan sulfate depresses the enzyme action. After this time the incubation mixture was transferred on the top of a Sephadex G-25 column, 1-2 cm × 35 cm. The sample vohime, Vs, must always follow the equation: Vs = ( K d ' - - K o " ) V I ; where Ko' and K d " are the distribution coefficients for the compounds separated. Kd' represents the distribution coefficient for the chondroitinBiochim. Biophys. dcta, 83 (1964) 137 -14o
138
PRELIMINARY NOTES
4-sulfate and chondroitin-6-sulfate "tetrasaccharides" and Ka" the distribution coefficient for the undegraded dermatan sulfate. V1 represents the internal volume of the gel matrix and was calculated as described 1~. The sample to be analyzed drained into the column by gravity and the column was washed with a constant flow rate of about I.O ml per 2 min with I.O M NaC1. Fractions of 1-2 ml were collected and the samples were analyzed for acetylhexosamines, uronic acid or total hexosamines. Compounds with molecular weights higher than 3500 are completely excluded from the gel bed of Sephadex G-25 used in this investigation 17, and appear therefore in an effluent volume equal to the void volume of the gel bed, V 0. Undegraded dermatan sulfate whose molecular weight lies above this order of magnitude appears therefore as the first peak of uronic-containing material in the effluent volume equal to Vo. This parameter was established experimentally as described TM. This fraction was analyzed for uronic acid by the borate method according to BITTER AND MEnd s as well as by the orcinol method TMand the original carbazol method of DISCHE~°. The ratio Aborate/ADtsehe w a s used to distinguish the dermatan sulfate and the other two isomers. This value for dermatan sulfate, and the chondroitin-4-sulfate and chondroitin-6-sulfate "tetrasaccharides" was 3.6o and 2.o6 respectively. Tetrasaccharides whose molecular weights lie below 35oo would emerge from the column when the effluent volume was equal to V o + (Ka' --Ka"). Fig. I shows the patterns of dermatan sulfate and the mixed sulfated tetrasaccharides in gel filtration. 7C
Chondroitin-6- sulfate t etrasac charide s
6C
5c 4C
u
3C
Dermatan sulfate
~6 2C
10 l~ 20 Volume of effluent (ml)
2~
Fig. I. P a t t e r n s of d e r m a t a n sulfate, a n d m i x e d c h o n d r o i t i n - 4 - s u l f a t e a n d c h o n d r o i t i n - 6 - s u l f a t e t e t r a s a c c h a r i d e s in gel filtration.
From each specimen containing the tetrasaccharides o.5-ml samples were taken for determination of acetylhexosamines by the method of REISSIG et al31. The standard acetylhexosamine curve was employed on N-acetyl-D-glucosamine, chromatographically homogeneous (kindly given by California Corporation for Biochemical Research, Lot 670731 ). Simultaneously, a further o.5-ml sample from the same fraction was taken for determination of total uronic acid TM. The amount of uronic acid present in the chondroitin-6-sulfate tetrasaccharides was calculated directly from the amount of acetylhexosamine found. The difference between the total amount of uronic acid found in the tetrasaccharides fraction and that due to the chondroitin6-sulfate fractions gives the amount of chondroitin 4-sulfate in ~g of uronic acid. Biochim. Biophys. Acta, 83 (1964) I 3 7 - I 4 O
PRELIMINARY NOTES
139
Total hexosamine determination can be used in place of total uronic acid, and the chondroitin 4-sulfates determined by subtracting the hexosamine due to chondroitin 6-sulfate, which is estimated as Morgan-Elson-positive acetylhexosamine. We wish to point out that, for this purpose, the chondroitin sulfate fraction must be freed of all other acid mucopolysaccharides before digestion. In the investigation here presented, the mixture of acid mucopolysaccharides was applied to a column of Sephadex A-25 as described previously 1 and fractionated by a modification 22 of the original method. Hyaluronic acid was completely eluted with 0.5 M NaC1. Heparan sulfates were eluted completely with 0.8-0. 9 M NaC1 zl. The chondroitin sulfates emerge from the column after applying 1.1-1.5 M NaC1 in o.oi N HC1. Dermatan sulfate so eluted showed no detectable amount of glucosamine, and its Aborate/ADisehe ratio was close to that given TM. Another point of importance is the composition of the "tetrasaccharides" obtained after hyahironidase digestion. Under the conditions here used, the TABLE I T H E AMOUNTS OF T H E I N D I V I D U A L ISOMERIC CHONDRO I T I N S U L F A T E S (AS J / g OF URONIC ACID) IN VARIOUS BIOLOGICAL MATERIALS
Chondroitin sulfate isomer found (..g J Or~in
Artificial mixture, ioo/~g C-4-S + ioo/~g DS Artificial mixture, ioo/~g C-4-S + IOO t*g C-6-S + 5o/~g DS H u m a n aorta, young, 2oo pg applied H u m a n aorta, adult, 2o0 # g applied H u m a n aorta, adult, atherosclerotic, 200/~g applied Bovine nasal cartilage, 2oo/~g applied Mouse skin, 200 # g applied
C-4-S
C-6-S
DS
91 89 ---186 --
-96 188 161 14o 9 24
ioi 46 7 29 48 -168
Abbreviations: C-4-S , chondroitin 4-sulfate; C-6-S, chondroitin 6-sulfate; DS, d e r m a t a n sulfate.
main oligosaccharide fraction was confirmed as tetrasaccharides. Gel filtration on larger columns (2 cm × 16o cm) showed that higher oligosaccharides (mainly hexaand octasaccharides) represent only lO-15 o//oof the total. The reducing value for the "tetrasaccharide" fraction obtained after digestion of pure standard chondroitin 6-sulfate was close to those calculated for pure tetrasaccharides. With this method it was possible to estimate the behavior of the chondroitin sulfate isomers in human aorta from young people and from adults with various degree of atherosclerosis 23 (Table I). Recoveries in all experiments were between 85 and 9 2 %.
Department of Biochemistry, University of Lodz, Lodz (Poland)
MARIAN SCHMIDT ANTONI DMOCHOWSKI
1 M. SCHMIDT, Biochim. Biophys. Acta, 63 (1962) 346. 2 G. S. BERENSON, S. ROSEMAN AND A. DORFMAN, Biochim. Biophys. Acta, 17 (1955) 75. s M. STACEY, Ann. Acad. Sei. Fennicae Ser. A II, 6o (1955) 262. 4 E. BUDDECKE, Physiol. Chem., 318 (196o) 335 S. SCHILLER, G. A. SLOVER AND A. DORFMAN, J. Biol. Chem., 236 (1961) 983. e M. B. MATHEWS, Biochim. Biophys. Acta, 48 (1961) 4o2. 7 G. S. BERENSON, Biochim. Biophys. Acta, 28 (1058) 176.
Bioehim. Biophys. Acla, 83 (1964) 137-14o
I40
PRELIMINARY
NOTES
8 K . MEYER, E . A . ])AX:IDSON, A. L I N K E R AND P. HOFFMAN, Biochim. Biophys. Acta, 21 ( [ 9 5 0 ) 5o(). 9 M. ]3. MATHEWS, Biochim. Biophys. Acla, 53 (1961) 5 0 9 • 10 R . K U H N , A. GAUHE AND H . H . BAER, Chem. Ber., 87 (1954) 1138. 11 R . \V. JEANLOZ AND M. TR~MEG~, Federation Proc., 15 (1950) 282. 12 J . i . C I F O N E L L I AND A . D O R F M A N , J. Biol. Chem., 23~ (1958) 1 i. la A . G. LLOYD, Nature, 183 (1959) 1o9. 14 1'. HO~'FMAN, A. LINKER AND K. MEYER, Bioehim. Biophys. Acta, 3 ° ( t 9 5 8 ) 184. 15 j . A. CIFONELLI, J . LUDOWIEG AND A. DORFMAN, J. Biol. Chem., 2 3 3 (1958) 54 I. In K . A. GRANATH AND P. FLODIN, Makromol. Chem., 48 (1901) i()o. 1'~ p . FLODIN AND K . A. GRANATH, Gel filtration applied to polymer fraclio~mtion, .';ymp. Makromol., Wiesbaden, 1959, W e i n h e i m . 18 T. BITTER AND H . M. MUIR, A n a l . Biochem., 4 (1(~02) 33 ° . 19 A. H . BROWN, Arch. Hiochem., 11 ( t 9 4 0 ) 200. 20 Z. DISCHE, J . Biol. Chem., 167 (1947) i89. 21 j . L. REISSlG, J . L. STROMINGER AND l.. F. LELOIR, .]. Biol. Chem., 2J 7 (1955) 959. 22 M. SCHMIDT, 1St Polish Biochem. Congr., Loda, 10, 1 9 6 3 . 2a M. SCHMIDT AND A. 1)MOCHOWSKI, ('omparative studies on acid mucopolysaccharides in young, adult and atherosclerolic human aortae, Geriatric .'~vmp. Polish Med. Noc., | ! ' a r s a w , 1963.
Received
July
Ist,
I9() 3
Biochim. HiophA,s. Acta, 83 (1904) I 3 7 14o
137
Preliminary Notes PN 81000
The determination of small quantities of isomeric chondroitin sulfates A new separation method for acid mucopolysaccharides was reported in a previous paper from this laboratory 1. Although a separation of extremely small amounts of connective-tissue mucopolysaccharides is possible by this method, the isomeric chondroitin sulfates remain unresolved. This deficiency also characterizes other presently available separation methods 2-5, although a partial separation of individual isomers m a y be obtained. Dermatan sulfate and chondroitin sulfate B are easily separated from the other two isomers b y electrophoresis at low pHe. Chondroitin 4-sulfate (chondroitin sulfate A) and dermatan sulfate (D) m a y be separated from one another by exploiting the different solubility of their Zn salts v. Chondroitin 4-sulfate and chondroitin 6-sulfate (chondroitin sulfate C) differ so little in their physical and chemical properties that a satisfactory separation of these isomers is difficults, or impossible. Lately MATHEWS9 described an elegant method for the determination of chondroitin sulfates of Type C, based on the observation that N-acetylhexosamines substituted only in position 6 behave normally in the Morgan-Elson reaction, while a substitution in position 4 causes an almost complete suppression of the color formation 1°-1~. The "acetylhexosamines" are released from chondroitin 4-sulfate and chondroitin 6-sulfate by the action of testicular hyaluronidase. This paper describes a procedure for the simultaneous determination of the isomeric chondroitin sulfates isolated from various biological materials. This procedure is founded on the following observations. (I) The known selective action of testicular hyaluronidase on chondroitin sulfates. Under the influence of this enzyme only the chondroitin-4-sulfate and chondroitin-6-sulfate isomers are split, leaving dermatan sulfate unaffected s, 14,15. (2) The different distribution coefficients, Ka, for the undigested dermatan sulfate molecule and the "tetrasaccharides" which are the main end products of chondroitin4-sulfate and chondroitin-6-sulfate degradation. (3) The anomalous behavior of Nacetylgalactosamine 4-sulfate in the Morgan-Elson reaction as mentioned above 9. I.O ml of the mixture containing only the isomeric chondroitin sulfates (50-500/~g of each isomer as ghicuronic acid) was mixed with an equal volume of citrate-phosphate buffer as described 9. I.O ml of a solution of testicular hyaluronidase in o.I M NaC1 containing lOO-6OO I.U. of the enzyme (Kinetin, Schering AG, Berlin, Germany) was added. This mixture was incubated over a 24-h period in glass-stoppered tubes at 38-4 o°. Although this reaction can be completed in a shorter time 9, a longer period was necessary because dermatan sulfate depresses the enzyme action. After this time the incubation mixture was transferred on the top of a Sephadex G-25 column, 1-2 cm × 35 cm. The sample vohime, Vs, must always follow the equation: Vs = ( K d ' - - K o " ) V I ; where Ko' and K d " are the distribution coefficients for the compounds separated. Kd' represents the distribution coefficient for the chondroitinBiochim. Biophys. dcta, 83 (1964) 137 -14o
138
PRELIMINARY NOTES
4-sulfate and chondroitin-6-sulfate "tetrasaccharides" and Ka" the distribution coefficient for the undegraded dermatan sulfate. V1 represents the internal volume of the gel matrix and was calculated as described 1~. The sample to be analyzed drained into the column by gravity and the column was washed with a constant flow rate of about I.O ml per 2 min with I.O M NaC1. Fractions of 1-2 ml were collected and the samples were analyzed for acetylhexosamines, uronic acid or total hexosamines. Compounds with molecular weights higher than 3500 are completely excluded from the gel bed of Sephadex G-25 used in this investigation 17, and appear therefore in an effluent volume equal to the void volume of the gel bed, V 0. Undegraded dermatan sulfate whose molecular weight lies above this order of magnitude appears therefore as the first peak of uronic-containing material in the effluent volume equal to Vo. This parameter was established experimentally as described TM. This fraction was analyzed for uronic acid by the borate method according to BITTER AND MEnd s as well as by the orcinol method TMand the original carbazol method of DISCHE~°. The ratio Aborate/ADtsehe w a s used to distinguish the dermatan sulfate and the other two isomers. This value for dermatan sulfate, and the chondroitin-4-sulfate and chondroitin-6-sulfate "tetrasaccharides" was 3.6o and 2.o6 respectively. Tetrasaccharides whose molecular weights lie below 35oo would emerge from the column when the effluent volume was equal to V o + (Ka' --Ka"). Fig. I shows the patterns of dermatan sulfate and the mixed sulfated tetrasaccharides in gel filtration. 7C
Chondroitin-6- sulfate t etrasac charide s
6C
5c 4C
u
3C
Dermatan sulfate
~6 2C
10 l~ 20 Volume of effluent (ml)
2~
Fig. I. P a t t e r n s of d e r m a t a n sulfate, a n d m i x e d c h o n d r o i t i n - 4 - s u l f a t e a n d c h o n d r o i t i n - 6 - s u l f a t e t e t r a s a c c h a r i d e s in gel filtration.
From each specimen containing the tetrasaccharides o.5-ml samples were taken for determination of acetylhexosamines by the method of REISSIG et al31. The standard acetylhexosamine curve was employed on N-acetyl-D-glucosamine, chromatographically homogeneous (kindly given by California Corporation for Biochemical Research, Lot 670731 ). Simultaneously, a further o.5-ml sample from the same fraction was taken for determination of total uronic acid TM. The amount of uronic acid present in the chondroitin-6-sulfate tetrasaccharides was calculated directly from the amount of acetylhexosamine found. The difference between the total amount of uronic acid found in the tetrasaccharides fraction and that due to the chondroitin6-sulfate fractions gives the amount of chondroitin 4-sulfate in ~g of uronic acid. Biochim. Biophys. Acta, 83 (1964) I 3 7 - I 4 O
PRELIMINARY NOTES
139
Total hexosamine determination can be used in place of total uronic acid, and the chondroitin 4-sulfates determined by subtracting the hexosamine due to chondroitin 6-sulfate, which is estimated as Morgan-Elson-positive acetylhexosamine. We wish to point out that, for this purpose, the chondroitin sulfate fraction must be freed of all other acid mucopolysaccharides before digestion. In the investigation here presented, the mixture of acid mucopolysaccharides was applied to a column of Sephadex A-25 as described previously 1 and fractionated by a modification 22 of the original method. Hyaluronic acid was completely eluted with 0.5 M NaC1. Heparan sulfates were eluted completely with 0.8-0. 9 M NaC1 zl. The chondroitin sulfates emerge from the column after applying 1.1-1.5 M NaC1 in o.oi N HC1. Dermatan sulfate so eluted showed no detectable amount of glucosamine, and its Aborate/ADisehe ratio was close to that given TM. Another point of importance is the composition of the "tetrasaccharides" obtained after hyahironidase digestion. Under the conditions here used, the TABLE I T H E AMOUNTS OF T H E I N D I V I D U A L ISOMERIC CHONDRO I T I N S U L F A T E S (AS J / g OF URONIC ACID) IN VARIOUS BIOLOGICAL MATERIALS
Chondroitin sulfate isomer found (..g J Or~in
Artificial mixture, ioo/~g C-4-S + ioo/~g DS Artificial mixture, ioo/~g C-4-S + IOO t*g C-6-S + 5o/~g DS H u m a n aorta, young, 2oo pg applied H u m a n aorta, adult, 2o0 # g applied H u m a n aorta, adult, atherosclerotic, 200/~g applied Bovine nasal cartilage, 2oo/~g applied Mouse skin, 200 # g applied
C-4-S
C-6-S
DS
91 89 ---186 --
-96 188 161 14o 9 24
ioi 46 7 29 48 -168
Abbreviations: C-4-S , chondroitin 4-sulfate; C-6-S, chondroitin 6-sulfate; DS, d e r m a t a n sulfate.
main oligosaccharide fraction was confirmed as tetrasaccharides. Gel filtration on larger columns (2 cm × 16o cm) showed that higher oligosaccharides (mainly hexaand octasaccharides) represent only lO-15 o//oof the total. The reducing value for the "tetrasaccharide" fraction obtained after digestion of pure standard chondroitin 6-sulfate was close to those calculated for pure tetrasaccharides. With this method it was possible to estimate the behavior of the chondroitin sulfate isomers in human aorta from young people and from adults with various degree of atherosclerosis 23 (Table I). Recoveries in all experiments were between 85 and 9 2 %.
Department of Biochemistry, University of Lodz, Lodz (Poland)
MARIAN SCHMIDT ANTONI DMOCHOWSKI
1 M. SCHMIDT, Biochim. Biophys. Acta, 63 (1962) 346. 2 G. S. BERENSON, S. ROSEMAN AND A. DORFMAN, Biochim. Biophys. Acta, 17 (1955) 75. s M. STACEY, Ann. Acad. Sei. Fennicae Ser. A II, 6o (1955) 262. 4 E. BUDDECKE, Physiol. Chem., 318 (196o) 335 S. SCHILLER, G. A. SLOVER AND A. DORFMAN, J. Biol. Chem., 236 (1961) 983. e M. B. MATHEWS, Biochim. Biophys. Acta, 48 (1961) 4o2. 7 G. S. BERENSON, Biochim. Biophys. Acta, 28 (1058) 176.
Bioehim. Biophys. Acla, 83 (1964) 137-14o
I40
PRELIMINARY
NOTES
8 K . MEYER, E . A . ])AX:IDSON, A. L I N K E R AND P. HOFFMAN, Biochim. Biophys. Acta, 21 ( [ 9 5 0 ) 5o(). 9 M. ]3. MATHEWS, Biochim. Biophys. Acla, 53 (1961) 5 0 9 • 10 R . K U H N , A. GAUHE AND H . H . BAER, Chem. Ber., 87 (1954) 1138. 11 R . \V. JEANLOZ AND M. TR~MEG~, Federation Proc., 15 (1950) 282. 12 J . i . C I F O N E L L I AND A . D O R F M A N , J. Biol. Chem., 23~ (1958) 1 i. la A . G. LLOYD, Nature, 183 (1959) 1o9. 14 1'. HO~'FMAN, A. LINKER AND K. MEYER, Bioehim. Biophys. Acta, 3 ° ( t 9 5 8 ) 184. 15 j . A. CIFONELLI, J . LUDOWIEG AND A. DORFMAN, J. Biol. Chem., 2 3 3 (1958) 54 I. In K . A. GRANATH AND P. FLODIN, Makromol. Chem., 48 (1901) i()o. 1'~ p . FLODIN AND K . A. GRANATH, Gel filtration applied to polymer fraclio~mtion, .';ymp. Makromol., Wiesbaden, 1959, W e i n h e i m . 18 T. BITTER AND H . M. MUIR, A n a l . Biochem., 4 (1(~02) 33 ° . 19 A. H . BROWN, Arch. Hiochem., 11 ( t 9 4 0 ) 200. 20 Z. DISCHE, J . Biol. Chem., 167 (1947) i89. 21 j . L. REISSlG, J . L. STROMINGER AND l.. F. LELOIR, .]. Biol. Chem., 2J 7 (1955) 959. 22 M. SCHMIDT, 1St Polish Biochem. Congr., Loda, 10, 1 9 6 3 . 2a M. SCHMIDT AND A. 1)MOCHOWSKI, ('omparative studies on acid mucopolysaccharides in young, adult and atherosclerolic human aortae, Geriatric .'~vmp. Polish Med. Noc., | ! ' a r s a w , 1963.
Received
July
Ist,
I9() 3
Biochim. HiophA,s. Acta, 83 (1904) I 3 7 14o