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A new method for the determination of N-sulfate in heparin and its analogs
ANALYTICAL
BIOCHEMISTRY
71, 46-52
(1976)
A New Method for the Determination of A/-Sulfate in Heparin and Its Analogs YUKO INOUE AND KINZO
NAGASAWA’
School of Pharmaceutical Sciences, Kitasato University, 9-1, Shirokane, 5 chome, Minato-ku, Tokyo 108, Japan Received June 10, 1975; accepted October 10, 1975 A new method for the determination of N-sulfate in heparin and its analogs is described. The method is based on the determination of inorganic sulfate liberated by deamination with nitrous acid. The accuracy, simplicity, and validity of this method are evaluated by comparing it with previous methods.
Heparin and heparitin sulfate are rare mucopolysaccharides containing N-sulfate groups in their structure. Recently, it has been reported that there are various types of heparin with different ratios of N-sulfate and N-acetyl content (1,2). It has also been known that blood anticoagulant activity of heparin is closely related to its N-sulfate content (3-5). Therefore, in these fields of research, an accurate determination of N-sulfate in samples is important. For the determination of N-sulfate, inorganic sulfate analysis (6) and calorimetric determination of dinitrophenylated (3) or trinitrophenylated amino groups (4) after acid hydrolysis of N-sulfate have been used. Another method consists of nitrous acid treatment and calorimetry of 25 anhydro-D-mannose residue with indole HCl(7,8). These methods of sulfate analysis have the unconquerable defect that the hydrolytic condition used is not strictly specific to N-sulfate. The latter two methods, colorimetric determination of free amino groups or 2,5-anhydro-D-mannose residues, also have a disadvantage that there is no universal and perfect standard for the determination of diverse N-sulfated mucopolysaccharides. In the course of research on the solvolytic N-desulfation of heparin (9), the authors have developed a simple and reliable method for the determination of N-sulfate in heparin. METHODS Materials
Heparins (Lot No. LPO30773 and LP011273) prepared from porcine intestinal mucosa were purchased from Cohelfred Laboratories, Chicago, I To whom correspondence
should be addressed 46
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction m any form reserved.
N-SULFATE
47
DETERMINATION
Ill. and had anticoagulant potency of 171.6 and 164.6 USP units, respectively. D-Glucose 6-sulfate (K salt) was obtained from Seikagaku Kogyo Co., Tokyo. Heparitin sulfate fraction (Ba salt), supplied by Upjohn Co., Kalamazoo, Mich., was fractionated on a column of Dowex-I (Cl-) to afford two purified fractions eluted with 1.25 M NaCl and 1.5 M NaCl(2). The sodium salt of 2-deoxy-2-sulfoamino-D-glucose was prepared by the method reported in our previous paper (10). N-Desulfated heparin was prepared by treating heparin (Lot No. LPO30773) in dimethyl sulfoxide containing 5% of water at 50°C for 90 min, and partially N-desulfated heparins were prepared by treating heparin (Lot No. LPOll273) in dimethyl sulfoxide containing 5% of water at 20°C for 5 to 120 min (9). N-Acetylation of heparin and N-desulfated heparins was carried out by the method of Danishefsky (9,l I). Determination Turbidimetry
of N-Sulfate of Inorganic
by Nitrous Acid Treatment Sulfate (Method Al2
followed
by
To 0.5 ml of a sample solution, 0.5 ml of 5% sodium nitrite and 0.5 ml of 33% acetic acid were added. After shaking, the solution was left standing at room temperature for 30 min. The solution was added to 4.5 ml of 3.8% trichloroacetic acid, shaken a little while, followed by the addition of 1.5 ml of the barium chloride-gelatin reagent (prepared by dissolving 1.0 g of gelatin and 0.5 g of barium chloride in 100 ml of water (I2)), and shaken again immediately. After standing for 20 min, turbidity of the solution was measured at 500 nm. Determination of Inorganic
of N-Sulfate by Acid Hydrolysis Sulfate (Method B)
followed
by Turbidimetry
Samples were hydrolyzed in 0.04 N HCl at 100°C for 2 hr, and the inorganic sulfate liberated was determined by Dodgson’s turbidimetry (6). Other Analytical
Methods
N-Acetyl content was estimated by the intensity of N-acetyl proton signals (6 2.05) relative to the total intensity of the signals due to two anomeric protons of the heparin disaccharide unit, heparin 6 5.24 (1H) and 6 5.36 (1H); N-acetylated heparin 6 5.36 (2H) in the NMR spectra. They were measured at 80°C with a JNM-PS-100 NMR spectrometer operated at 100 MHz in deuterium oxide containing sodium 4,4-dimethyl4-silapentane-1-sulfonate as an internal standard. * In the absence of 33% acetic acid, desulfation of sulfoamino groups similarly proceeds in 3.8% trichloroacetic acid (4.5 ml) for 30 min. and successively the barium chloridegelatin reagent is added to the reaction mixture to determine the liberated inorganic sulfate.
48
INOUE
AND
NAGASAWA
Total sulfate in samples was analyzed by Dodgson’s turbidimetry of inorganic sulfate liberated by hydrolysis in 1 N HCl at 100°C for 5 hr. 2-Amino-2-deoxy-D-glucose in 2-deoxy-2-sulfoamino-D-glucose, heparin, and heparitin sulfate was determined according to the method of Dische and Borenfreund (7) using 2-amino-2-deoxy-D-glucose as a standard, except that the reaction time for deamination of both heparin and heparitin sulfate was extended to 90 min to obtain a maximum coloration with amino sugar. Determination of 2-deoxy-2-sulfoamino-D-glucose in heparin and heparitin sulfate was carried out by the method of Cifonelli (13) using sodium salt of 2-deoxy-2-sulfoamino-D-glucose as a standard, except that the reaction time for deamination was reduced to 3 hr to obtain a constant value of sulfoamino sugar. RESULTS
AND DISCUSSION
The present method for analysis of N-sulfate (Method A) is based on a turbidimetric determination of inorganic sulfate (6) liberated by deamination of sulfoamino sugar with nitrous acid (7). The 30-min incubation in nitrous acid is somewhat shorter than the optimal time indicated in (8). However, it has been confirmed that a+constant N-sulfate value is obtained by incubation of heparin during 30 to 60 min after the start of reaction. Because no step to destroy nitrite is in the proposed method, conditions for the desulfation of sulfoamino groups are kept during the course of analysis. As shown in Table 1, the sulfur content in 2-deoxy-2-sulfoamino-Dglucose determined by Method A was 10.97% which is in good agreement with the value (10.8%) determined under the condition of total sulfate analysis. The 2-deoxy-2-sulfoamino-D-glucose used in this experiment is a crystalline sodium salt which migrates uniformly on paper electrophoresis, and its slightly lower sulfur content is possibly due to the presence of crystal water hard to remove. Under the same condition (Method A), D-glucose 6-sulfate gave no inorganic sulfate, but 10.95% of sulfur was quantified from an equimolar mixture of 2-deoxy-2-sulfoamino-D-glucose and D-glucose 6-sulfate. These results show that Method A is specific to iv-sulfate quantification. On the other hand, turbidimetric sulfate analysis after hydrolysis of the equimolar mixture in 0.04 N HCI at 100°C for 2 hr (Method B) gave the value of 13.20% which is much higher than the N-sulfate content calculated for the mixture. This indicates that hydrolysis of O-sulfate and N-sulfate should occur under the conditions used in Method B. Determination of N-sulfate in heparin by Method A showed a sulfur content of 4.24%, which corresponds to 0.79 mollmol hexosamine, assuming that the polysaccharide chain in heparin consists of repeated disaccharide units of hexuronic acid and hexosamine (see the footnote in Table 1). Content of N-acetyl groups in heparin and IV-acetylated heparin
N-SULFATE
DETERMINATION
49
was determined from NMR spectral measurement, and their content was 0.11 and 0.18 mol/mol hexosamine, respectively (Table 2). The difference between their N-acetyl contents, 0.07 mol/mol hexosamine, corresponds to the content of free amino groups present in heparin. This value agrees with the content of TNP-amino groups in heparin as indicated by the ratio of coloration (CH&,,), 0.076: 1, obtained by N-trinitrophenylation of heparin and N-desulfated heparin which was reported previously (9). Provided no amino group of amino acids is present in heparin, a value of 0.18 mol means the total molar amount of 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-o-glucose per mole of hexosamine, and the value seems appropriate for 0.79 mol of N-sulfate content determined with heparin by Method A. On the other hand, determination of N-sulfate TABLE COMPARISON
1
OF INORGANIC SULFATE LIBERATED ACID HYDROLYSIS OF N-SULFATED
BY NITRITE COMPOUNDS
TREATMENT OR
N-Sulfate Method A Compound 2-Deoxy-2-sulfoamino-oglucose” Equimolar mixture of 2deoxy-2-suffoamino-oglucose and D-glucose 6-sulfate Heparin* Heparitin sulfate 1.5 M NaCl fraction 1.25 M NaCl fraction
Method B
(S%)
(moBc
(S%)
(mol)’
10.97
0.96
10.83
0.95
10.95 4.24
0.96 0.79
13.20 5.30
4.03 2.83
0.65 0.38
5.70 2.55
Total sulfate N-Acetyl (mob
(S%)
(mol)
1.16 1.00
12.44
2.34
0.11
0.91 0.35
10.19 5.11
I .63 0.69
0.24 0.66
n Calculated S: I I .40%. b Lot No. LPO30773. r Molar number of N-sulfate in heparin, heparitin sulfate, or N-desulfated heparin in Tables 1 and 2 was calculated according to the following equations: x is obtained from sxx C,,H,,_,0,0NH,-dCOCH3),Na
+ (SO,Na),
x 100 = Total sulfate (S%)
and then x’ is obtained from s XX’ C,,H,,-,O,~H,-,(COCH,),Na
+ (SO&&
x 100 = N-Sulfate (S%)
where x is molar number of total sulfate per mole of hexosamine; x’ is molar number of N-sulfate per mole of hexosamine; y is molar number of N-acetyl group per mole of hexosamine, where N-acetyl group is determined by the NMR measurement.
50
INOUE
AND TABLE
NAGASAWA 2
CONTENTS OF N-SULFATE AND N-ACETYL GROUP DETERMINED ON HEPARIN, N-DESULFATED HEPARIN, AND THEIR N-ACETYLATED PRODUCTS
Compound” Heparin (1) N-Acetylated N-Desulfated N-Acetylated N-desulfated (l)-(3)
(4-Q)
heparin (2) heparin (3) product of heparin (4)
N-Sulfate (mol)b
N-Acetyl (moi)
0.79 0.05
0.11 0.18 -
0.74
0.89 0.71
D Modified heparins (2-4) are those derived from heparin (I) (lot No. LPO30773). * See the footnote c in Table f .
by Method B gave a value of 5.30% which is much higher than that obtained by Method A, suggesting a partial hydrolysis of O-sulfate in heparin. In the case of heparitin sulfate shown in Table 1, determination of N-sulfate in the fraction eluted with 1.5 M NaCl gave a higher value with Method B and a reasonable value with Method A, taking its N-acetyl content into consideration, On the other hand, the fraction eluted with 1.25 M NaCl having a lower sulfur content and a higher N-acetyl content gave a higher N-sulfate value with Method A than that with Method B. N-Desulfated heparin prepared by solvolysis in dimethyi sulfoxide containing 5% of water at 50°C for 90 min (9) was analyzed by Method A to give the N-sulfate content of 0.05 mol/mol hexosamine which indicates removal of 94% of N-sulfate in the original heparin (Table 2). The difference between the N-acetyl contents of the N-acetylated product of N-desulfated heparin and N-acetylated heparin is 0.71 mol which should correspond to the amount of free amino groups liberated by solvolytic Ndesulfation, and the value agrees fairly well with the difference (0.74 mol) in both N-sulfate contents in heparin and N-desulfated heparin determined by Method A. The data obtained by Method A on partially N-desulfated heparins and their ZV-acetylated products prepared by solvolytic N-desulfation accompanied with N-acetylation are summarized in Table 3. It is notable that the sample containing about a half molar N-acetyl group per mole of hexosamine gave a reasonable N-sulfate value. The amount of iv-sulfate in 2-deoxy-2-sulfoamino-D-glucose was determined by the nitriteaindole HCI method (7) using 2-amino-2-deoxy-Dglucose as a standard, and the value obtained was markedly lower than that calculated (Table 4). Furthermore, the amount of N-sulfate in heparin and heparitin sulfate fractions was determined by the method of butyl
N-SULFATE
51
DETERMINATION TABLE
3
IV-SULFATE CONTENTDETERMINEDON PARTIALLYN-DESULFATED AND THEIR N-ACETYLATED PRODUCTS
Compound” Heparin Heparin partially N-desulfated at 20°C for 5 min at 20°C for IO min at 20°C for 20 min at 20°C for 60 min at 20°C for 120 min N-Acetylated heparin N-Acetylated product of heparin partially N-desulfated at 20°C for 5 min at 20°C for 10 min at 20°C for 20 min at 20°C for 60 min at 20°C for 120 min
HEPARINS
Total sulfate (S%)
N-Sulfate 6%)
N-Acetyl (mol/mol of hexosamine)
12.11
3.96
0.12
11.48 II.35 11.09 10.84 10.66 11.91
3.61 3.45 3.44 2.85 2.50 3.80
0.14
11.45 11.36 11.33 11.13 10.95
3.35 3.30 3.22 2.56 2.37
0.17 0.20 0.24 0.37 0.49
a All the compounds tabulated in this table are heparin of lot No. LPO11273 and those derived from it.
nitrite sindole HCl(13) which is said to be specific to N-sulfoamino groups, besides the conventional method just described using 2-deoxy-2-sulfoamino-D-glucose and 2-amino-2-deoxy-D-glucose as a standard. Two Nsulfate values in heparin obtained by these methods were diverse as shown in Table 4. Assuming that all of the hexosamine in heparin or heparitin sulfate is N-sulfated and/or N-acetylated, a reasonable N-sulfate *ralue was obtained by the method of nitrite*indole HCl, while the butyl nitrite*indole HCl method gave a rather higher value. In the case of heparitin sulfate, the values obtained were also diverse, and were generally higher than those expected from their N-acetyl content. The results shown in Table 4 suggest that both methods, consisting of two successive reactions -deamination with nitrous acid or with butyl nitrite and formation of a chromogen with indole in HCl-have a serious defect which is supposedly due to unavailability of a true and proper standard for them.3 Judging from the experimental results obtained, the proposed method based on the determination of inorganic sulfate liberated from only sulfo3 A standard of choice often used in the estimation of N-sulfate groups is heparin of known N-sulfate or N-acetyl content (13). Several investigators have noted the possibility of discrepant results when using 2-amino-2-deoxy-D-glucose as a standard for the estimation of N-sulfated compounds by the nitrite. indole reaction.
52
INOUE AND NAGASAWA TABLE
DETERMINATION
4
OF 2-DEOXY-2-SULFOAMINO-D-GLUCOSE IN HEPARIN AND HEPARITIN SULFATE BY NITROUS ACID OR BUTYL NITRITE TREATMENT FOLLOWED BY INDOLE HCl METHOD
N-Sulfate (molb)
Compound 2-Deoxy-2-sulfoamino-D-glucose Heparin” Heparitin sulfate 1.5 M NaCI fraction I.25 M NaCl fraction
Butyl nitrite .indole HCl
N-Acetyl (mol)
0.69 0.87
1.08
0.11
0.78 0.44
0.71 0.41
0.24 0.66
Nitrite.indole HCI
a Lot No. LPO30773. b Molar number of N-sulfate per mole of hextisamine was calculated from the amount of 2-amino-2-deoxy-D-glucose or 2-deoxy-2-sulfoamino-D-glucose determined, assuming that the polysaccharide chain in heparin consists of repeated disaccharide units of hexuronic acid and hexosamine including N-acetylated hexosamine (0. II mol).
amino groups by deaminative desulfation with nitrous acid seems to be excellent in its simplicity and validity, although there is a limited precision due to the use of turbidimetry. A further advantage of this method lies in being insensitive to contaminant carbohydrates, nucleic acids, and other substances which may interfere with the former procedure. The authors suggest that the merit of this method lies in its direct analysis of N-sulfate groups and the fact that it provides another procedure which is both simple and relatively reliable for study of an important class of compounds. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Cifonelli, J. A., and King, J. (1970) Curbohyd. Res. 12, 391-402. Cifonelli, J. A., and King J. (1973) Biochim. Biophys. Acta 320, 331-340. Foster, A. B., Martlew, E. F., and Stacey. M. (1953) Chem. Ind. (London) 899-900. Yoshizawa, Z., Kotoku, T., Yamauchi, F., and Matsuno, M. (1967)Biochim. Eiophys. Acta 141, 358-365. Cifonelli, J. A. (1974) Carbohyd. Res. 37, 145-154. Dodgson, K. S. (1961) Eiochem. J. 78, 312-319; (1962) ibid. 84, 106-110. Dische, Z., and Borenfreund, E. (1950) J. Biol. Chem. 184, 517-522. Lagunoff, D., and Warren, G. (1962) Arch. Eiochem. Eiophys. 99, 396-400. Inoue, Y., and Nagasawa, K. (1976) Carbohyd. Res. 46, 87-95. Inoue, Y., and Nagasawa, K. (1973) J. Org. Chem. 38, 1810-1813. Danishefsky, I., Eiber, H. B., and Carr, J. J. (1960) Arch. E&hem. Eiophys. 90, 114-121. Kawai, Y.. Seno, N., and Anno, K. (1969)Anal. Eiochem. 32, 314-321. Cifonelli, J. A., and King. J. (1972) Carbohyd. Res. 21, 173-186.
BIOCHEMISTRY
71, 46-52
(1976)
A New Method for the Determination of A/-Sulfate in Heparin and Its Analogs YUKO INOUE AND KINZO
NAGASAWA’
School of Pharmaceutical Sciences, Kitasato University, 9-1, Shirokane, 5 chome, Minato-ku, Tokyo 108, Japan Received June 10, 1975; accepted October 10, 1975 A new method for the determination of N-sulfate in heparin and its analogs is described. The method is based on the determination of inorganic sulfate liberated by deamination with nitrous acid. The accuracy, simplicity, and validity of this method are evaluated by comparing it with previous methods.
Heparin and heparitin sulfate are rare mucopolysaccharides containing N-sulfate groups in their structure. Recently, it has been reported that there are various types of heparin with different ratios of N-sulfate and N-acetyl content (1,2). It has also been known that blood anticoagulant activity of heparin is closely related to its N-sulfate content (3-5). Therefore, in these fields of research, an accurate determination of N-sulfate in samples is important. For the determination of N-sulfate, inorganic sulfate analysis (6) and calorimetric determination of dinitrophenylated (3) or trinitrophenylated amino groups (4) after acid hydrolysis of N-sulfate have been used. Another method consists of nitrous acid treatment and calorimetry of 25 anhydro-D-mannose residue with indole HCl(7,8). These methods of sulfate analysis have the unconquerable defect that the hydrolytic condition used is not strictly specific to N-sulfate. The latter two methods, colorimetric determination of free amino groups or 2,5-anhydro-D-mannose residues, also have a disadvantage that there is no universal and perfect standard for the determination of diverse N-sulfated mucopolysaccharides. In the course of research on the solvolytic N-desulfation of heparin (9), the authors have developed a simple and reliable method for the determination of N-sulfate in heparin. METHODS Materials
Heparins (Lot No. LPO30773 and LP011273) prepared from porcine intestinal mucosa were purchased from Cohelfred Laboratories, Chicago, I To whom correspondence
should be addressed 46
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction m any form reserved.
N-SULFATE
47
DETERMINATION
Ill. and had anticoagulant potency of 171.6 and 164.6 USP units, respectively. D-Glucose 6-sulfate (K salt) was obtained from Seikagaku Kogyo Co., Tokyo. Heparitin sulfate fraction (Ba salt), supplied by Upjohn Co., Kalamazoo, Mich., was fractionated on a column of Dowex-I (Cl-) to afford two purified fractions eluted with 1.25 M NaCl and 1.5 M NaCl(2). The sodium salt of 2-deoxy-2-sulfoamino-D-glucose was prepared by the method reported in our previous paper (10). N-Desulfated heparin was prepared by treating heparin (Lot No. LPO30773) in dimethyl sulfoxide containing 5% of water at 50°C for 90 min, and partially N-desulfated heparins were prepared by treating heparin (Lot No. LPOll273) in dimethyl sulfoxide containing 5% of water at 20°C for 5 to 120 min (9). N-Acetylation of heparin and N-desulfated heparins was carried out by the method of Danishefsky (9,l I). Determination Turbidimetry
of N-Sulfate of Inorganic
by Nitrous Acid Treatment Sulfate (Method Al2
followed
by
To 0.5 ml of a sample solution, 0.5 ml of 5% sodium nitrite and 0.5 ml of 33% acetic acid were added. After shaking, the solution was left standing at room temperature for 30 min. The solution was added to 4.5 ml of 3.8% trichloroacetic acid, shaken a little while, followed by the addition of 1.5 ml of the barium chloride-gelatin reagent (prepared by dissolving 1.0 g of gelatin and 0.5 g of barium chloride in 100 ml of water (I2)), and shaken again immediately. After standing for 20 min, turbidity of the solution was measured at 500 nm. Determination of Inorganic
of N-Sulfate by Acid Hydrolysis Sulfate (Method B)
followed
by Turbidimetry
Samples were hydrolyzed in 0.04 N HCl at 100°C for 2 hr, and the inorganic sulfate liberated was determined by Dodgson’s turbidimetry (6). Other Analytical
Methods
N-Acetyl content was estimated by the intensity of N-acetyl proton signals (6 2.05) relative to the total intensity of the signals due to two anomeric protons of the heparin disaccharide unit, heparin 6 5.24 (1H) and 6 5.36 (1H); N-acetylated heparin 6 5.36 (2H) in the NMR spectra. They were measured at 80°C with a JNM-PS-100 NMR spectrometer operated at 100 MHz in deuterium oxide containing sodium 4,4-dimethyl4-silapentane-1-sulfonate as an internal standard. * In the absence of 33% acetic acid, desulfation of sulfoamino groups similarly proceeds in 3.8% trichloroacetic acid (4.5 ml) for 30 min. and successively the barium chloridegelatin reagent is added to the reaction mixture to determine the liberated inorganic sulfate.
48
INOUE
AND
NAGASAWA
Total sulfate in samples was analyzed by Dodgson’s turbidimetry of inorganic sulfate liberated by hydrolysis in 1 N HCl at 100°C for 5 hr. 2-Amino-2-deoxy-D-glucose in 2-deoxy-2-sulfoamino-D-glucose, heparin, and heparitin sulfate was determined according to the method of Dische and Borenfreund (7) using 2-amino-2-deoxy-D-glucose as a standard, except that the reaction time for deamination of both heparin and heparitin sulfate was extended to 90 min to obtain a maximum coloration with amino sugar. Determination of 2-deoxy-2-sulfoamino-D-glucose in heparin and heparitin sulfate was carried out by the method of Cifonelli (13) using sodium salt of 2-deoxy-2-sulfoamino-D-glucose as a standard, except that the reaction time for deamination was reduced to 3 hr to obtain a constant value of sulfoamino sugar. RESULTS
AND DISCUSSION
The present method for analysis of N-sulfate (Method A) is based on a turbidimetric determination of inorganic sulfate (6) liberated by deamination of sulfoamino sugar with nitrous acid (7). The 30-min incubation in nitrous acid is somewhat shorter than the optimal time indicated in (8). However, it has been confirmed that a+constant N-sulfate value is obtained by incubation of heparin during 30 to 60 min after the start of reaction. Because no step to destroy nitrite is in the proposed method, conditions for the desulfation of sulfoamino groups are kept during the course of analysis. As shown in Table 1, the sulfur content in 2-deoxy-2-sulfoamino-Dglucose determined by Method A was 10.97% which is in good agreement with the value (10.8%) determined under the condition of total sulfate analysis. The 2-deoxy-2-sulfoamino-D-glucose used in this experiment is a crystalline sodium salt which migrates uniformly on paper electrophoresis, and its slightly lower sulfur content is possibly due to the presence of crystal water hard to remove. Under the same condition (Method A), D-glucose 6-sulfate gave no inorganic sulfate, but 10.95% of sulfur was quantified from an equimolar mixture of 2-deoxy-2-sulfoamino-D-glucose and D-glucose 6-sulfate. These results show that Method A is specific to iv-sulfate quantification. On the other hand, turbidimetric sulfate analysis after hydrolysis of the equimolar mixture in 0.04 N HCI at 100°C for 2 hr (Method B) gave the value of 13.20% which is much higher than the N-sulfate content calculated for the mixture. This indicates that hydrolysis of O-sulfate and N-sulfate should occur under the conditions used in Method B. Determination of N-sulfate in heparin by Method A showed a sulfur content of 4.24%, which corresponds to 0.79 mollmol hexosamine, assuming that the polysaccharide chain in heparin consists of repeated disaccharide units of hexuronic acid and hexosamine (see the footnote in Table 1). Content of N-acetyl groups in heparin and IV-acetylated heparin
N-SULFATE
DETERMINATION
49
was determined from NMR spectral measurement, and their content was 0.11 and 0.18 mol/mol hexosamine, respectively (Table 2). The difference between their N-acetyl contents, 0.07 mol/mol hexosamine, corresponds to the content of free amino groups present in heparin. This value agrees with the content of TNP-amino groups in heparin as indicated by the ratio of coloration (CH&,,), 0.076: 1, obtained by N-trinitrophenylation of heparin and N-desulfated heparin which was reported previously (9). Provided no amino group of amino acids is present in heparin, a value of 0.18 mol means the total molar amount of 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-o-glucose per mole of hexosamine, and the value seems appropriate for 0.79 mol of N-sulfate content determined with heparin by Method A. On the other hand, determination of N-sulfate TABLE COMPARISON
1
OF INORGANIC SULFATE LIBERATED ACID HYDROLYSIS OF N-SULFATED
BY NITRITE COMPOUNDS
TREATMENT OR
N-Sulfate Method A Compound 2-Deoxy-2-sulfoamino-oglucose” Equimolar mixture of 2deoxy-2-suffoamino-oglucose and D-glucose 6-sulfate Heparin* Heparitin sulfate 1.5 M NaCl fraction 1.25 M NaCl fraction
Method B
(S%)
(moBc
(S%)
(mol)’
10.97
0.96
10.83
0.95
10.95 4.24
0.96 0.79
13.20 5.30
4.03 2.83
0.65 0.38
5.70 2.55
Total sulfate N-Acetyl (mob
(S%)
(mol)
1.16 1.00
12.44
2.34
0.11
0.91 0.35
10.19 5.11
I .63 0.69
0.24 0.66
n Calculated S: I I .40%. b Lot No. LPO30773. r Molar number of N-sulfate in heparin, heparitin sulfate, or N-desulfated heparin in Tables 1 and 2 was calculated according to the following equations: x is obtained from sxx C,,H,,_,0,0NH,-dCOCH3),Na
+ (SO,Na),
x 100 = Total sulfate (S%)
and then x’ is obtained from s XX’ C,,H,,-,O,~H,-,(COCH,),Na
+ (SO&&
x 100 = N-Sulfate (S%)
where x is molar number of total sulfate per mole of hexosamine; x’ is molar number of N-sulfate per mole of hexosamine; y is molar number of N-acetyl group per mole of hexosamine, where N-acetyl group is determined by the NMR measurement.
50
INOUE
AND TABLE
NAGASAWA 2
CONTENTS OF N-SULFATE AND N-ACETYL GROUP DETERMINED ON HEPARIN, N-DESULFATED HEPARIN, AND THEIR N-ACETYLATED PRODUCTS
Compound” Heparin (1) N-Acetylated N-Desulfated N-Acetylated N-desulfated (l)-(3)
(4-Q)
heparin (2) heparin (3) product of heparin (4)
N-Sulfate (mol)b
N-Acetyl (moi)
0.79 0.05
0.11 0.18 -
0.74
0.89 0.71
D Modified heparins (2-4) are those derived from heparin (I) (lot No. LPO30773). * See the footnote c in Table f .
by Method B gave a value of 5.30% which is much higher than that obtained by Method A, suggesting a partial hydrolysis of O-sulfate in heparin. In the case of heparitin sulfate shown in Table 1, determination of N-sulfate in the fraction eluted with 1.5 M NaCl gave a higher value with Method B and a reasonable value with Method A, taking its N-acetyl content into consideration, On the other hand, the fraction eluted with 1.25 M NaCl having a lower sulfur content and a higher N-acetyl content gave a higher N-sulfate value with Method A than that with Method B. N-Desulfated heparin prepared by solvolysis in dimethyi sulfoxide containing 5% of water at 50°C for 90 min (9) was analyzed by Method A to give the N-sulfate content of 0.05 mol/mol hexosamine which indicates removal of 94% of N-sulfate in the original heparin (Table 2). The difference between the N-acetyl contents of the N-acetylated product of N-desulfated heparin and N-acetylated heparin is 0.71 mol which should correspond to the amount of free amino groups liberated by solvolytic Ndesulfation, and the value agrees fairly well with the difference (0.74 mol) in both N-sulfate contents in heparin and N-desulfated heparin determined by Method A. The data obtained by Method A on partially N-desulfated heparins and their ZV-acetylated products prepared by solvolytic N-desulfation accompanied with N-acetylation are summarized in Table 3. It is notable that the sample containing about a half molar N-acetyl group per mole of hexosamine gave a reasonable N-sulfate value. The amount of iv-sulfate in 2-deoxy-2-sulfoamino-D-glucose was determined by the nitriteaindole HCI method (7) using 2-amino-2-deoxy-Dglucose as a standard, and the value obtained was markedly lower than that calculated (Table 4). Furthermore, the amount of N-sulfate in heparin and heparitin sulfate fractions was determined by the method of butyl
N-SULFATE
51
DETERMINATION TABLE
3
IV-SULFATE CONTENTDETERMINEDON PARTIALLYN-DESULFATED AND THEIR N-ACETYLATED PRODUCTS
Compound” Heparin Heparin partially N-desulfated at 20°C for 5 min at 20°C for IO min at 20°C for 20 min at 20°C for 60 min at 20°C for 120 min N-Acetylated heparin N-Acetylated product of heparin partially N-desulfated at 20°C for 5 min at 20°C for 10 min at 20°C for 20 min at 20°C for 60 min at 20°C for 120 min
HEPARINS
Total sulfate (S%)
N-Sulfate 6%)
N-Acetyl (mol/mol of hexosamine)
12.11
3.96
0.12
11.48 II.35 11.09 10.84 10.66 11.91
3.61 3.45 3.44 2.85 2.50 3.80
0.14
11.45 11.36 11.33 11.13 10.95
3.35 3.30 3.22 2.56 2.37
0.17 0.20 0.24 0.37 0.49
a All the compounds tabulated in this table are heparin of lot No. LPO11273 and those derived from it.
nitrite sindole HCl(13) which is said to be specific to N-sulfoamino groups, besides the conventional method just described using 2-deoxy-2-sulfoamino-D-glucose and 2-amino-2-deoxy-D-glucose as a standard. Two Nsulfate values in heparin obtained by these methods were diverse as shown in Table 4. Assuming that all of the hexosamine in heparin or heparitin sulfate is N-sulfated and/or N-acetylated, a reasonable N-sulfate *ralue was obtained by the method of nitrite*indole HCl, while the butyl nitrite*indole HCl method gave a rather higher value. In the case of heparitin sulfate, the values obtained were also diverse, and were generally higher than those expected from their N-acetyl content. The results shown in Table 4 suggest that both methods, consisting of two successive reactions -deamination with nitrous acid or with butyl nitrite and formation of a chromogen with indole in HCl-have a serious defect which is supposedly due to unavailability of a true and proper standard for them.3 Judging from the experimental results obtained, the proposed method based on the determination of inorganic sulfate liberated from only sulfo3 A standard of choice often used in the estimation of N-sulfate groups is heparin of known N-sulfate or N-acetyl content (13). Several investigators have noted the possibility of discrepant results when using 2-amino-2-deoxy-D-glucose as a standard for the estimation of N-sulfated compounds by the nitrite. indole reaction.
52
INOUE AND NAGASAWA TABLE
DETERMINATION
4
OF 2-DEOXY-2-SULFOAMINO-D-GLUCOSE IN HEPARIN AND HEPARITIN SULFATE BY NITROUS ACID OR BUTYL NITRITE TREATMENT FOLLOWED BY INDOLE HCl METHOD
N-Sulfate (molb)
Compound 2-Deoxy-2-sulfoamino-D-glucose Heparin” Heparitin sulfate 1.5 M NaCI fraction I.25 M NaCl fraction
Butyl nitrite .indole HCl
N-Acetyl (mol)
0.69 0.87
1.08
0.11
0.78 0.44
0.71 0.41
0.24 0.66
Nitrite.indole HCI
a Lot No. LPO30773. b Molar number of N-sulfate per mole of hextisamine was calculated from the amount of 2-amino-2-deoxy-D-glucose or 2-deoxy-2-sulfoamino-D-glucose determined, assuming that the polysaccharide chain in heparin consists of repeated disaccharide units of hexuronic acid and hexosamine including N-acetylated hexosamine (0. II mol).
amino groups by deaminative desulfation with nitrous acid seems to be excellent in its simplicity and validity, although there is a limited precision due to the use of turbidimetry. A further advantage of this method lies in being insensitive to contaminant carbohydrates, nucleic acids, and other substances which may interfere with the former procedure. The authors suggest that the merit of this method lies in its direct analysis of N-sulfate groups and the fact that it provides another procedure which is both simple and relatively reliable for study of an important class of compounds. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Cifonelli, J. A., and King, J. (1970) Curbohyd. Res. 12, 391-402. Cifonelli, J. A., and King J. (1973) Biochim. Biophys. Acta 320, 331-340. Foster, A. B., Martlew, E. F., and Stacey. M. (1953) Chem. Ind. (London) 899-900. Yoshizawa, Z., Kotoku, T., Yamauchi, F., and Matsuno, M. (1967)Biochim. Eiophys. Acta 141, 358-365. Cifonelli, J. A. (1974) Carbohyd. Res. 37, 145-154. Dodgson, K. S. (1961) Eiochem. J. 78, 312-319; (1962) ibid. 84, 106-110. Dische, Z., and Borenfreund, E. (1950) J. Biol. Chem. 184, 517-522. Lagunoff, D., and Warren, G. (1962) Arch. Eiochem. Eiophys. 99, 396-400. Inoue, Y., and Nagasawa, K. (1976) Carbohyd. Res. 46, 87-95. Inoue, Y., and Nagasawa, K. (1973) J. Org. Chem. 38, 1810-1813. Danishefsky, I., Eiber, H. B., and Carr, J. J. (1960) Arch. E&hem. Eiophys. 90, 114-121. Kawai, Y.. Seno, N., and Anno, K. (1969)Anal. Eiochem. 32, 314-321. Cifonelli, J. A., and King. J. (1972) Carbohyd. Res. 21, 173-186.