- Email: [email protected]
Spectrophotometric determination of acid mucopolysaccharides
ANALYTICAL
BIOCHEMISTRY
51, 274-279 (1973)
Spectrophotometric
Determination
of
Acid
Mucopolysaccharides A. H. WARD1 Biochemical
AR’D
G. A. MICHOS
Research Depadment, Warren State Hospitul, Warren, Pe?lnsulvaGa 16565
Received July 19, 1972; accepted August, 30, 1972 The present report describes a novel spectrophotometric method for the quantitative determination of acid mucopolysaccharides based on the interaction of these macromolecules with the zirconyl ion. The method is simple, accurate, and involves the determination of the acid mucopolysaccharide molecules rather than their hydrolytic components (as in the case of existing methods of analysis). Substances, which are normally present in the acid mucopolysaccharide preparations (such as protein, glycoprotein, and nucleic acid), do not interfere with the determination.
The quantitative determination of acid mucopolysaccharides in various biological preparations presents a problem frequently encountered in the biochemical investigation of t’hese macromolecules. The problem arises primarily from the lack of a suitable method for the separation and quantitative determination of these substances. The amount of mucopolysaccharides (in any given sample) is usually calculated from the amount of glucuronic acid and/or hexosamine which are determined spectrophotometrically by the carbazole-sulfuric acid (1) and Elson-Morgan reaction (2), respectively. Results obtained by these methods, however, are of little value when the acid mucopolysaccharide preparations are contaminated with substances which contain the component being determined, such as glycoprotein, (which contains hexosamine) or glucuronic acid conjugates (which contain glucuronic acid). In addition, the assumption that mucopolysaccharides (even those which belong to the same group) have similar composition is erroneous and often leads to unreliable results. In 1956, Hahn and Gaginiski (3)) used mandelic acid for the quantitative precipitation of zirconium from hot and strongly acidic media. In 1959, Wardi (4), investigated the interaction of other hydroxy carboxylic acids with the zirconium ion. Recent studies in our laborat,ory have shown that acid mucopolysac274 Copyright ,@ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACID
MUCOPOLYSACCHARIDE
DETERMINATION
275
charides, which also contain both hydroxyl and carboxyl groups, interact with zirconium to form insoluble complexes. The present report describes a new approach for the quantitative determination of acid mucopolysaccharide based on their interaction with zirconyl ion. The procedure involves the precipitation of acid mucopolysaccharides from dilute hydrochloric acid medium with zirconyl chloride solution, and the subsequent spectrophotometric determination of excess zirconyl ion in the supernatant fluid with alizarin red S reagent (5). EXPERIMENTAL
Reagents 1. Zirconyl chloride solution. One gram of zirconyl chloride (ZrOCl,, 8H,O, Fisher Scientific Co.), dissolved in 1 liter of distilled water containing 2 ml of hydrochloric acid. 2. Alizarin red S. Three twentieth gram of alizarin red S (Sigma Chemical Co.) was dissolved in 30 ml of hot water, filtered, and diluted to 100 ml. 3. Zinc chloride solution. Zinc metal (assay 99.9%, Baker-analyzed reagent) was dissolved in a few milliliters of 6 N HCl and diluted to 500 ml with distilled water. 4. Xylenol orange. Xylenol orange (BDG Chemicals) 0.01% aqueous solution. 5. Catechol violet. Catechol violet (BDG Chemicals) 0.01% aqueous solution. 6. Chondroitin sulfate type C. Miles Laboratory special grade preparation. Procedure Spectrophotometric determination of acid mucopolysaccharide. To 2 ml of acid mucopolysaccharide solution (containing 0.5 mg sample in 1 ml) in 0.035 M HCl, 1 ml of zirconyl chloride solution was added. The precipitate formed was allowed to coagulate and filtered. An aliquot (1 ml) of the filtrate was mixed with 0.4 ml of concentrated HCl, 4 ml water, and 1 ml alizarin red S reagent and placed in a bath of boiling water for 21$$min. The solution was cooled to room temperature, diluted to 10 ml with distilled water and the absorbance was measured at 550 mp. A control containing 2 ml of water, instead of acid mucopolysaccharide solution, and a blank solution containing 1 ml of alizarin red S reagent but no zirconyl chloride or acid mucopolysaccharide, were also prepared. The percentage of acid mucopolysaccharide in an unknown sample was calculated as follows:
276
WARD1
AND
MICHOS
% AMPS = [C,(OD, - OD,)/C,(OD,
- OO,)] x 100 x P,
where C, = concentration of standard acid mucopolysaccharide in milligrams per millilit~er. C, = concentration of unknown in milligrams per milliliter. OD,, OD,, and 011, refer to the optical densities of control, standard acid mucopolysaccharide, and unknown samples, respectively P = percentage of acid mucopolysaccharides in the standard. Standardization of zirconyl chloride solution. The concentration of the zirconyl chloride solution was determined by titration with EDTA; an aliquot (1 ml) of zirconyl chloride was mixed with 1 ml of concentrated HCl, boiled, and diluted to 100 ml with distilled water. Catechol violet (8 drops) was added and the solution was titrated with EDTA solution. The EDTA solution, in turn, was standardized against zinc chloride solution; an aliquot (25 ml) of zinc chloride solution was diluted to 50 ml with 0.1 M acetate buffer of pH 5. Xylenol orange (8 drops) was added and the solution was titrated with EDTA (7). Composition of acid mucopolysaccharide zirconium complexes. To 3 ml acid mucopolysaccharide in 0.035 M HCl, 1 ml of standard zirconyl chloride solution was added. The precipitate formed was filtered and 3 ml of the filtrate was boiled with 1 ml of concentrated HCl. The solution was diluted to 100 ml with water and titrated with standard EDTA as described. To determine the amount of zirconium used by acid mucopolysaccharide in the sample, the volume of standard EDTA used was subtracted from the volume of EDTA required to titrate a similar sample but without any acid mucopolysaccharide added. RESULTS
AND
DISCUSSION
Zirconyl ions form colored complexes with alizarin red S in hydrochloric acid solution. Heating the zirconyl solution with HCl at 100°C for 21/, min in the presence of alizarin red S produces a red color which exhibits an absorption maxima at 550 mp. Under these conditions the optical density of the color is proportional to the zirconyl ion concentration. When acid mucopolysaccharide (AMPS) is also present in the reaction mixture the zirconyl ion reacts with it to form insoluble complexes. This reaction, therefore, withdraws zirconyl ions from the solutions and results in a reduction in the color of the solution. In this procedure, proteins, glycoproteins, gum arabic, and nucleic acids do not interfere with AMPS determination. Nucleic acid (if present) is usually precipitated upon acidification of the sample solution. The pre-
ACID
MUCOPOLYSACCHARIDE
DETERMINATION
277
cipitate is removed by filtration through a thin Celite bed prior to the AMPS determination. Other substances which form complexes with zirconium in acidic media, and strong oxidizing agents which destroy the alizarin red S. reagent, interfere with these determinations. These substances, however, are not likely to be present in AMPS preparations. Phosphate in highly impure preparations may be removed by precipitation with magnesia solution. Analysis of biological preparations. To test the accuracy of the method, the following commercial preparations were analyzed by the carbazolesulfuric acid, and the spectrophotometric methods, using glucuronolactone and CSC (Miles Laboratory) as standards, respectively: (a) Chondroitin sulfate sodium, bovine nasal septa (Mann Research Laboratories). (b) Chondroitin sulfate sodium bovine nasal septa (General Biochemicals) . (c) Chondroitin sulfate purified, glucuronic acid 22.1% (General Biochemicals) . Samples of these preparations were dried to constant weight. under diminished pressure over P,O5 at 65°C prior to analysis. Results of these analyses are shown in Table 1. The amount of AMPS in commercial preparations determined by the spectrophotometric method are within 0.5% of the values obtained by the carbazole-H,S04 reaction. In these calculations the value of (P) was 86.86 which represents the percentage of sulfated AMPS in the Miles Laboratories preparation as determined by the carbazole reaction. For the determination of other types of AMPS (such as hyaluronic acid, heparin, etc.) corresponding standard should be used in the respective determinations. Composition of Zr-ZAMP complexes. To 4.06 mg of CSC in 3 ml of 0.035 M HCl, 1 ml of standard zirconyl chloride solution (0.0176 M) was added and the mixture was centrifuged. A 3-ml aliquot of the supernatant fluid was boiled with 1 ml of concentrated HCl, diluted to 100 ml with water and titrated with standard EDTA solution (0.010 M) using catechol violet as indicator. To determine the amount of zirconium used for the precipitation of CSC, the volume of EDTA (0.8 ml) consumed, was subtracted from the (2.4 ml) EDTA volume to titrate 3 ml of zirconium solution prepared similarly but without CSC added. The difference (1.6 ml) corresponds to 0.016 mmole of zirconium. Assuming that the molecular weight of CSC is 50,000, the number of millimoles of CSC in the solution is 0.81 X 10-4, or that each mole of CSC has reacted with 200 moles of Zr.
formula,
b Calculated
u For
from
percentage
0.3373 0.3360 0.3400 0.2093
Weight (w/ml)
Mucopolysaccharide
multiplying
see text.
acid)
of Acid
Mann, nasal septa GBI, nasal septa GBI (22.1 To hexuronic CYC (Miles Lab.)
Sample
Determination
nm
550
hexuronic
0.083 0.065 0.135 0.144
OD
acid
in Commercial
C
ODn
by the ratio:
2.4319 2.5574 2.0814 3.2885
OD,.
OD,-
TABLE 1 Preparations
JfII, Hexuronic
M, CS disaccharide
66.32 69.74 56.76 -
y. AMPS present method
acid
by the Carbazole
=
500.398 194.14
25.87 27.15 21.83 34.80
= 2 5775. .
66.68 69.98 56.26 86.86
% AMPSb carbazole
the Spectrophotometric
y0 Hexuronic acid carbazole
and
-0.36 -0.24 +0.50
To Difference in AMPS values
Methods $
5 s z z! ,o
$ El
ACID
MUCOPOLTSACCHARIDE
279
DETERMINATION
ACKNOWLEDGMENTS This work 05932-07 PC.
was supported
in part
by NIH,
NINDS
Research
Grant
5-ROl-
REFERENCES 1. DISCHE, 2. 3. 4.
5. 6. 7.
Z. (1947) J. Biol. Chem. 16’7, 189. ELSON, L. A., AND MORGAN, W. T. J. (1933) Biochem. J. 27, 1824. HAHN, R. B., AND BAGINISKI, E. S. (1956) Anal. Chem. Acta 14, 45. WARDI, A. H. (1959) Ph.D. thesis, Wayne State University. HAHN, R. B. (1967) in Treatise on Analytical Chemistry (I. M. Kolthoff P. T. Elving, eds.), Part II, Vol. 5, pp. 61-138, Interscience, New York. BARAN, V., AND TYMPL, M. (1964) Coil. Czech. Chem. Commun. 29, 2250. KORBL, J., AND PRIBIL, R. (1956) Chem. Anal. 45, 102.
and
BIOCHEMISTRY
51, 274-279 (1973)
Spectrophotometric
Determination
of
Acid
Mucopolysaccharides A. H. WARD1 Biochemical
AR’D
G. A. MICHOS
Research Depadment, Warren State Hospitul, Warren, Pe?lnsulvaGa 16565
Received July 19, 1972; accepted August, 30, 1972 The present report describes a novel spectrophotometric method for the quantitative determination of acid mucopolysaccharides based on the interaction of these macromolecules with the zirconyl ion. The method is simple, accurate, and involves the determination of the acid mucopolysaccharide molecules rather than their hydrolytic components (as in the case of existing methods of analysis). Substances, which are normally present in the acid mucopolysaccharide preparations (such as protein, glycoprotein, and nucleic acid), do not interfere with the determination.
The quantitative determination of acid mucopolysaccharides in various biological preparations presents a problem frequently encountered in the biochemical investigation of t’hese macromolecules. The problem arises primarily from the lack of a suitable method for the separation and quantitative determination of these substances. The amount of mucopolysaccharides (in any given sample) is usually calculated from the amount of glucuronic acid and/or hexosamine which are determined spectrophotometrically by the carbazole-sulfuric acid (1) and Elson-Morgan reaction (2), respectively. Results obtained by these methods, however, are of little value when the acid mucopolysaccharide preparations are contaminated with substances which contain the component being determined, such as glycoprotein, (which contains hexosamine) or glucuronic acid conjugates (which contain glucuronic acid). In addition, the assumption that mucopolysaccharides (even those which belong to the same group) have similar composition is erroneous and often leads to unreliable results. In 1956, Hahn and Gaginiski (3)) used mandelic acid for the quantitative precipitation of zirconium from hot and strongly acidic media. In 1959, Wardi (4), investigated the interaction of other hydroxy carboxylic acids with the zirconium ion. Recent studies in our laborat,ory have shown that acid mucopolysac274 Copyright ,@ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACID
MUCOPOLYSACCHARIDE
DETERMINATION
275
charides, which also contain both hydroxyl and carboxyl groups, interact with zirconium to form insoluble complexes. The present report describes a new approach for the quantitative determination of acid mucopolysaccharide based on their interaction with zirconyl ion. The procedure involves the precipitation of acid mucopolysaccharides from dilute hydrochloric acid medium with zirconyl chloride solution, and the subsequent spectrophotometric determination of excess zirconyl ion in the supernatant fluid with alizarin red S reagent (5). EXPERIMENTAL
Reagents 1. Zirconyl chloride solution. One gram of zirconyl chloride (ZrOCl,, 8H,O, Fisher Scientific Co.), dissolved in 1 liter of distilled water containing 2 ml of hydrochloric acid. 2. Alizarin red S. Three twentieth gram of alizarin red S (Sigma Chemical Co.) was dissolved in 30 ml of hot water, filtered, and diluted to 100 ml. 3. Zinc chloride solution. Zinc metal (assay 99.9%, Baker-analyzed reagent) was dissolved in a few milliliters of 6 N HCl and diluted to 500 ml with distilled water. 4. Xylenol orange. Xylenol orange (BDG Chemicals) 0.01% aqueous solution. 5. Catechol violet. Catechol violet (BDG Chemicals) 0.01% aqueous solution. 6. Chondroitin sulfate type C. Miles Laboratory special grade preparation. Procedure Spectrophotometric determination of acid mucopolysaccharide. To 2 ml of acid mucopolysaccharide solution (containing 0.5 mg sample in 1 ml) in 0.035 M HCl, 1 ml of zirconyl chloride solution was added. The precipitate formed was allowed to coagulate and filtered. An aliquot (1 ml) of the filtrate was mixed with 0.4 ml of concentrated HCl, 4 ml water, and 1 ml alizarin red S reagent and placed in a bath of boiling water for 21$$min. The solution was cooled to room temperature, diluted to 10 ml with distilled water and the absorbance was measured at 550 mp. A control containing 2 ml of water, instead of acid mucopolysaccharide solution, and a blank solution containing 1 ml of alizarin red S reagent but no zirconyl chloride or acid mucopolysaccharide, were also prepared. The percentage of acid mucopolysaccharide in an unknown sample was calculated as follows:
276
WARD1
AND
MICHOS
% AMPS = [C,(OD, - OD,)/C,(OD,
- OO,)] x 100 x P,
where C, = concentration of standard acid mucopolysaccharide in milligrams per millilit~er. C, = concentration of unknown in milligrams per milliliter. OD,, OD,, and 011, refer to the optical densities of control, standard acid mucopolysaccharide, and unknown samples, respectively P = percentage of acid mucopolysaccharides in the standard. Standardization of zirconyl chloride solution. The concentration of the zirconyl chloride solution was determined by titration with EDTA; an aliquot (1 ml) of zirconyl chloride was mixed with 1 ml of concentrated HCl, boiled, and diluted to 100 ml with distilled water. Catechol violet (8 drops) was added and the solution was titrated with EDTA solution. The EDTA solution, in turn, was standardized against zinc chloride solution; an aliquot (25 ml) of zinc chloride solution was diluted to 50 ml with 0.1 M acetate buffer of pH 5. Xylenol orange (8 drops) was added and the solution was titrated with EDTA (7). Composition of acid mucopolysaccharide zirconium complexes. To 3 ml acid mucopolysaccharide in 0.035 M HCl, 1 ml of standard zirconyl chloride solution was added. The precipitate formed was filtered and 3 ml of the filtrate was boiled with 1 ml of concentrated HCl. The solution was diluted to 100 ml with water and titrated with standard EDTA as described. To determine the amount of zirconium used by acid mucopolysaccharide in the sample, the volume of standard EDTA used was subtracted from the volume of EDTA required to titrate a similar sample but without any acid mucopolysaccharide added. RESULTS
AND
DISCUSSION
Zirconyl ions form colored complexes with alizarin red S in hydrochloric acid solution. Heating the zirconyl solution with HCl at 100°C for 21/, min in the presence of alizarin red S produces a red color which exhibits an absorption maxima at 550 mp. Under these conditions the optical density of the color is proportional to the zirconyl ion concentration. When acid mucopolysaccharide (AMPS) is also present in the reaction mixture the zirconyl ion reacts with it to form insoluble complexes. This reaction, therefore, withdraws zirconyl ions from the solutions and results in a reduction in the color of the solution. In this procedure, proteins, glycoproteins, gum arabic, and nucleic acids do not interfere with AMPS determination. Nucleic acid (if present) is usually precipitated upon acidification of the sample solution. The pre-
ACID
MUCOPOLYSACCHARIDE
DETERMINATION
277
cipitate is removed by filtration through a thin Celite bed prior to the AMPS determination. Other substances which form complexes with zirconium in acidic media, and strong oxidizing agents which destroy the alizarin red S. reagent, interfere with these determinations. These substances, however, are not likely to be present in AMPS preparations. Phosphate in highly impure preparations may be removed by precipitation with magnesia solution. Analysis of biological preparations. To test the accuracy of the method, the following commercial preparations were analyzed by the carbazolesulfuric acid, and the spectrophotometric methods, using glucuronolactone and CSC (Miles Laboratory) as standards, respectively: (a) Chondroitin sulfate sodium, bovine nasal septa (Mann Research Laboratories). (b) Chondroitin sulfate sodium bovine nasal septa (General Biochemicals) . (c) Chondroitin sulfate purified, glucuronic acid 22.1% (General Biochemicals) . Samples of these preparations were dried to constant weight. under diminished pressure over P,O5 at 65°C prior to analysis. Results of these analyses are shown in Table 1. The amount of AMPS in commercial preparations determined by the spectrophotometric method are within 0.5% of the values obtained by the carbazole-H,S04 reaction. In these calculations the value of (P) was 86.86 which represents the percentage of sulfated AMPS in the Miles Laboratories preparation as determined by the carbazole reaction. For the determination of other types of AMPS (such as hyaluronic acid, heparin, etc.) corresponding standard should be used in the respective determinations. Composition of Zr-ZAMP complexes. To 4.06 mg of CSC in 3 ml of 0.035 M HCl, 1 ml of standard zirconyl chloride solution (0.0176 M) was added and the mixture was centrifuged. A 3-ml aliquot of the supernatant fluid was boiled with 1 ml of concentrated HCl, diluted to 100 ml with water and titrated with standard EDTA solution (0.010 M) using catechol violet as indicator. To determine the amount of zirconium used for the precipitation of CSC, the volume of EDTA (0.8 ml) consumed, was subtracted from the (2.4 ml) EDTA volume to titrate 3 ml of zirconium solution prepared similarly but without CSC added. The difference (1.6 ml) corresponds to 0.016 mmole of zirconium. Assuming that the molecular weight of CSC is 50,000, the number of millimoles of CSC in the solution is 0.81 X 10-4, or that each mole of CSC has reacted with 200 moles of Zr.
formula,
b Calculated
u For
from
percentage
0.3373 0.3360 0.3400 0.2093
Weight (w/ml)
Mucopolysaccharide
multiplying
see text.
acid)
of Acid
Mann, nasal septa GBI, nasal septa GBI (22.1 To hexuronic CYC (Miles Lab.)
Sample
Determination
nm
550
hexuronic
0.083 0.065 0.135 0.144
OD
acid
in Commercial
C
ODn
by the ratio:
2.4319 2.5574 2.0814 3.2885
OD,.
OD,-
TABLE 1 Preparations
JfII, Hexuronic
M, CS disaccharide
66.32 69.74 56.76 -
y. AMPS present method
acid
by the Carbazole
=
500.398 194.14
25.87 27.15 21.83 34.80
= 2 5775. .
66.68 69.98 56.26 86.86
% AMPSb carbazole
the Spectrophotometric
y0 Hexuronic acid carbazole
and
-0.36 -0.24 +0.50
To Difference in AMPS values
Methods $
5 s z z! ,o
$ El
ACID
MUCOPOLTSACCHARIDE
279
DETERMINATION
ACKNOWLEDGMENTS This work 05932-07 PC.
was supported
in part
by NIH,
NINDS
Research
Grant
5-ROl-
REFERENCES 1. DISCHE, 2. 3. 4.
5. 6. 7.
Z. (1947) J. Biol. Chem. 16’7, 189. ELSON, L. A., AND MORGAN, W. T. J. (1933) Biochem. J. 27, 1824. HAHN, R. B., AND BAGINISKI, E. S. (1956) Anal. Chem. Acta 14, 45. WARDI, A. H. (1959) Ph.D. thesis, Wayne State University. HAHN, R. B. (1967) in Treatise on Analytical Chemistry (I. M. Kolthoff P. T. Elving, eds.), Part II, Vol. 5, pp. 61-138, Interscience, New York. BARAN, V., AND TYMPL, M. (1964) Coil. Czech. Chem. Commun. 29, 2250. KORBL, J., AND PRIBIL, R. (1956) Chem. Anal. 45, 102.
and