ANALYTICAL
BIOCHEMISTRY
78, 112- 118 (1977)
Determination of Sulfate after Chromatography
Toluidine Blue Complex Formation
HELENA
Departamento
de Bioquimica
and
B. NADER AND CARL P. DIETRICH
e Farmacologia,
S&o Paula,
Escola Paulista
SP., Brazil
de Medicina,
C.P. 20.372,
Received July 13, 1976: accepted October 19, 1976
A rapid and simple method for the determination of sulfate involving a complex
formation between inorganic sulfate and the dye, toluidine blue 0, after
chromatography, is presented. The method can be used for the determination of
sulfate in the presence of interfering ions such as phosphate and citrate. Most
of the ions have a different chromatographic migration in the solvent system
employed. An added advantage is the measurement of the labile sulfate of
mucopolysaccharides with accuracy.
Several analytical methods have been developed for determination
of
the sulfate of mucopolysaccharides
(MPS) (l-9). Most of these methods
are based on the release of inorganic sulfate from MPS by acid hydrolysis
followed by measurement of the liberated sulfate with benzidine (l-3),
barium chloride (4,5), barium chloride-gelatin
(6- 8)) or chloranilate (9).
These procedures are based on the precipitation of sulfate and measurement of the precipitate after either a calorimetric reaction or a direct uv
spectrophotometry.
The content of sulfate groups in MPS has also been
determined by conductimetry
(lo), oxidation-combustion
for S analysis
(1 l), and gas-liquid chromatography
(12).
Labile sulfate (N-sulfate) groups which are present in heparin and
heparitin sulfates’ have been determined
by the following methods:
treatment of the mucopolysaccharides
with nitrous acid followed by
inorganic sulfate determination
(4) or calorimetric
determination
of the
2,5-anhydro-D-mannose
with indole-HCl
(13,14); acid hydrolysis of the
sulfoamino groups of the MPS and inorganic sulfate analysis (6,7) or
calorimetric determination
of the free amino groups formed (15); a modification of the nitrous acid treatment of the MPS and turbidimetry
of the
inorganic sulfate (16). All these methods require large amounts of MPS
and, besides, the liberated sulfate is measured by indirect means which
is not entirely satisfactory.
In this paper we report the determination
of sulfate by the complex
formation
between the inorganic sulfate (generated either after acid
r Heparitin sulfate is also known as heparan sulfate, chondroitin sulfate A and C as
chondroitin 4-sulfate and chondroitin 6-sulfate, chondroitin sulfate B as dermatan sulfate.
112
Copyright
All rights
0 1977 by Academic Press. Inc.
of reproduction
in any form reserved.
ISSN ooO3-2697
DETERMINATION
OF SULFATE
113
hydrolysis or enzymatic degradation) and the dye toluidine blue 0. Staining with toluidine blue 0 is performed after separation of the inorganic
sulfate from other contaminating
ions by chromatography.
MATERIALS
AND METHODS
Chemicals. Commercial heparin preparations were kindly supplied by
the UpJohn Company (Kalamazoo,
Mich.) and Lederle Laboratories
(Division of American Cyannamid; Pearl River, N. Y.). Heparin hexa-,
tetra-, and disaccharides, glucosamine 2,6-bissulfate, and glucosamine
N-sulfate were prepared by enzymatic degradation as previously described
(17,18). Chondroitin
sulfates A, B, and C were purchased from Miles
Laboratories
(Elkhart, Ind.). Heparitin sulfates A, B, C, and D were
prepared as previously described (19). Sulfated disaccharides from
chondroitin
sulfates A and C were prepared by enzymatic degradation
as described by Saito et al. (8). Reagent-grade sodium sulfate or potassium
sulfate (E. Merck AG., Darmstadt, Germany) was used as the standard
sulfate. Toluidine
blue 0 was purchased from Fisher Scientific Co.
(Fairlawn, N. J.) or Matheson Coleman & Bell, Matheson Co., Inc.
(Norwood, Ohio; East Rutherford, N. J.). The dye obtained from B.D.H.
(British Drug House, England) and E. Merck AG. (Darmstadt, Germany)
produced unsatisfactory results. Chemicals other than those listed above
were all reagent grade.
Glassware. Glassware was cleaned with nitric acid. Mixtures containing sulfuric acid were avoided. Micropipets were of the Lang-Levy
constriction type (H. E. Pedersen, Denmark).
Chromatography.
Sheets of Whatman No. 1 paper (46 x 57 cm) from
W. & R. Ralston Ltd. (England) were used for descending chromatography. Ascending thin-layer
chromatography
was performed on 20
x 20-cm Eastman
Chromagram
sheets (silica gel without fluorescent
indicator) from Distillation
Products Industries (Division of Eastman
Kodak Company, Rochester, N. Y.).
Densitometry.
Densitometry
was performed with the Model G Computer microdensitometer
from Canalco (Rockville, Md.).
Preparation of the toluidine blue 0 reagent. Toluidine blue 0 (500 mg)
was dissolved in 500 ml of absolute ethanol. The solution was then filtered
through filter paper to remove undissolved particles of the dye and
stored in a closed container.
Other methods. Amino sugars were measured after acid hydrolysis
(4 M HCl for 6 hr at 1OO’C) by a modified Elson-Morgan
reaction (20).
Procedure for the sulfate analysis after acid hydrolysis. Labile and total
sulfate are measured after hydrolysis of the mucopolysaccharides
(20- 100 pg) in 0.04 M HCl for 2 hr at 100°C and 8 M HCl for 6 hr at
lOo”C, respectively,
in sealed capillary tubes with a final volume of
114
NADER
AND
DIETRICH
20 ~1. Appropriate standards containing 2-40 pg of sodium sulfate (or
potassium sulfate) and a reagent blank are subjected to the same procedure. After hydrolysis, the solution is transferred to a microtube
(0.4-cm diameter x 5-cm height), and the capillary tube is washed with
50 ~1 of distilled water. The combined solutions are evaporated to
dryness under vacuum over NaOH. The residue is resuspended in 50 ~1
of distilled water and dried again. The residue is then resuspended in
20 pliter of 0.5 M Na2C03, spotted on Whatman No. 1 paper chromatogram, and subjected to descending chromatography
in isobutyric acid:
1 M NHIOH
(5:3, v/v) for 6 to 8 hr or until the solvent moves about
15 cm from the origin of the chromatogram.
There should be a distance
of 3 cm between each spot. The chromatogram is then dried and stained
by dipping it into the toluidine blue 0 solution for lo-15 min. This time
period may vary according to the source of the dye. The paper is then
destained with absolute ethanol by the dipping procedure (three washes,
lo-15 min each with occasional agitation or until the background
appears clear and homogenous). A control of nonhydrolyzed
material
should be run in the chromatogram
because MPS preparations often
contain inorganic sulfate contamination.
The amounts of inorganic sulfate in the spots are measured by densitometry at 600 nm. An alternate procedure is to cut the chromatogram
containing the stained spot of inorganic sulfate, immerse it in a solution
of 2 M N&SO, for 2 hr with agitation, and measure the resulting solution
by spectrophotometry
at 630 nm. Appropriate blanks of the same dimensions and cut from the same stained chromatogram
are run throughout
this procedure. The error in both procedures is on the order of 4.5%.
The same sequence of steps is also applied after ascending chromatography on thin-layer chromatograms with the same solvent system used in
the paper chromatography
system.
RESULTS
AND DISCUSSION
The standard curve of sulfate as determined by the toluidine blue
method is shown in Fig. 1. The curve is linear, at least up to 20 ,ug of
potassium sulfate. The mean error obtained for each point is approximately 24.5%. This method detects as little as 2 pg of potassium sulfate.
Several known by-products of hydrolysis were tested as possible
interfering agents of the sulfate determination.
Among the sugars, glucose,
glucosamine,
glucuronic acid, and IV-acetylglucosamine
had no disturbing effects on the determination
(up to 20 times the concentration
of sulfate). Among the ions, 0.5 pequiv of each of the chloride salts of
Na+, K+, NH4+, Mg2+, Ca2+, Ba2+, and Fe3+ and 1.0 pequiv of each of
the sodium salts of acetate, bicarbonate, carbonate, chloride, nitrate, and
nitrite had no detectable complex formation with the dye and did not
DETERMINATION
115
OF SULFATE
150-
Q
;
IOO-
CT
lW
2
2
G
z
50-
0
I,,,,,,,,,
4
6
12
16
20
FIG. 1. The optical density of the chromatograms for the sulfate-toluidine
blue complex. Absorbance was measured after densitometry of the chromatogram at 600 nm (for
details, see Materials and Methods).
interfere with the determinations.
Besides sulfate, the anions citrate,
oxalate, hydrogen phosphate, dihydrogen phosphate, and pyrophosphate
show bluish spots of various intensities in the chromatogram.
Nevertheless, these ions migrate more rapidly than the inorganic sulfate (Table 1).
Table 2 shows the labile and total sulfate content of mucopolysaccharides measured by the toluidine blue method. The values obtained agree
TABLE
CHROMATOGRAPHIC
MIGRATION
I
OF DIFFERENT
ANIONP
Anion
Chromatographic
migration (Rise,)*
Sulfate
Citrate
Hydrogen phosphate
Dihydrogen phosphate
Pyrophosphate
1.0
1.41
1.43
1.35
1.11
a About 20 4 of a 0.025 N solution of the different anions (Na+ salt) were applied to
Whatman No. 1 paper and subjected to chromatography as described in Materials
and Methods.
*R iso. - the migration relative to sulfate.
116
NADER AND DIETRICH
TABLE
DETERMINATION
2
OF LABILE
AND TOTAL
SULFATE
IN MUCOPOLYSACCHARIDES
AND THEIR
DEGRADATION
PRODUCTS
Molar ratios to hexosamine
Mucopolysaccharide
Labile sulfate
Total sulfate
Heparin
Glucosamine N-sulfate
Glucosamine 2,6-bissulfate
Heparin trisulfated disaccharide
Heparin disulfated disaccharide
Heparin tetrasaccharide
Heparitin sulfate A
Heparitin sulfate B
Heparitin sulfate C
Heparitin sulfate D
Heparitin sulfate (crude)
Chondroitin sulfate A
Chondroitin sulfate B
Chondroitin sulfate C
Chondroitin sulfate A disaccharide
Chondroitin sulfate C disaccharide
1.05
1.04
0.93
0.97
0.93
0.97
0.04
0.35
0.94
1.08
0.74
-
2.61
0.93
1.83
3.08
1.82
2.46
0.45
0.95
2.02
2.62
1.55
0.97
1.01
1.07
0.95
1.03
-
very well with those reported for this class of compounds but obtained
by different procedures.
Furthermore,
it gives accurate values of labile sulfate for the MPS
and their degradation products such as glucosamine, 2,6-bissulfate, and
the sulfated di- and tetrasaccharides.
The reason for this is the finding
that the sulfated by-products of the reaction either remain at the origin
of the chromatogram
or have a different chromatographic
migration than
inorganic sulfate. Heparin and its enzymatic degradation products are
completely separated from the liberated inorganic sulfate after mild acid
hydrolysis. A possible source of error in the determination
of sulfate by
the present method may be the presence of reaction by-products with
the same chromatographic
migration of inorganic sulfate. For instance, the
4-sulfated disaccharide formed by the action of chondroitinase AC upon
chondroitin sulfate A has the same chromatographic
migration of inorganic sulfate in the solvent system used. This problem can be overcome
by using electrophoresis instead of chromatography
for the quantitation
of inorganic sulfate (21).
The method is also very useful to determine directly the sulfate content of biological fluids in the presence of other interfering ions, as exemplified in Fig. 2 which shows the chromatogram of a urine sample. Note
that the inorganic sulfate is completely
separated from the inorganic
DETERMINATION
OF SULFATE
117
i
FIG. 2. Sulfate determination in urine after paper chromatography and toluidine blue
0 staining: 5, 10, and 20 pg of N&SO., (l-3); 10, 20, and 30 ~1 of normal urine sample
(4-6); 15. 30, and 60 pg of NaH,PO,.H,O (7-9).
phosphate which can also be measured by the present method. This
experiment also illustrates the sensibility of the method; 10 pliter or less
of urine is sufficient for the determination.
This method is even simpler
than that of Wainer and Koch (22) for determination
of sulfate in urine
using barium chloranilate.
The continuous use of the method described in this paper has proven
it to be rapid, reproducible, accurate, and very sensitive for the characterization of mucopolysaccharides.
ACKNOWLEDGMENTS
We wish to thank Dr. Sonia M. C. Dietrich for helpful criticisms and suggestions. In
this research we were aided by grants from FAPESP (Funda&o de Amparo a Pesquisa
do Estado de Ssio Paulo), CNPq (Conselho National do Desenvolvimen to Cientifico e
Tecnologico), and FINEP (Financiadora de Estudos e Projetos).
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