ANALYTICAL
BIOCHEMISTRY
(1991)
197,157-162
Measurement of Uranic Acids without from Neutral Sugars Tullia
M. C. C. Filisetti-Cozzi*
and Nicholas
C. Carpita?,’
*Department0 de Alimentos e Nutri@o Experimental da Faculdade Postal 30786, CEP 01051, S6o Paul0 SP, Brazil; and TDepartment Purdue University, West Lafayette, Indiana 47907
Received
February
Carbazole was introduced for the quantitative determination of uranic acid in concentrated sulfuric acid (1,2). In the presence of other sugars, particularly hexoses, substantial interference that compromised specificity was observed, and the method was modified in
0003-2697/91 Copyright All rights
de Ci&cias Farmachticas, Universidade of Botany and Plant Pathology,
S&o Paulo,
27,199l
Replacement of carbazole with meta-hydroxydiphenyl greatly improves the determination of uranic acids in the presence of neutral sugars by preventing substantially, but not completely, the browning that occurs during the heating of sugars in concentrated sulfuric acid and avoiding the formation of additional interference by the carbazole reagent (Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54,484489). However, interference is still substantial when uranic acids are determined in the presence of excess neutral sugar, particularly because of the browning that occurs during the first heating before addition of the diphenyl reagent. The browning can be essentially eliminated by addition of sulfamate to the reaction mixture (Galambos, J. T. (1967) And. Biochem. 19, 119132). Although others have reported that sulfamate and the diphenyl reagent were incompatible, we find that a small amount of sulfamate suppresses color production by a 20-fold excess of some neutral sugars without substantial sacrifice of the sensitive detection of uranic acids by the diphenyl reagent. Sodium tetraborate is required for the detection of D-mannuronic acid and enhances color production by D-ghCUrOniC acid. We propose this modified sulfamatelm-hydroxydiphenyl assay as a rapid and reliable means for the assay of uranic acids, particularly when present in much smaller o 1991 Academic press, IIW. amOUntS than neutral sugars.
1 To whom
Interference
correspondence
should
$3.00 0 1991 by Academic Press, of reproduction in any form
be addressed.
several ways to reduce color production by neutral sugars. Gregory (3) and Bitter and Muir (4) observed that addition of borate to the carbazole reaction increased color production by certain uranic acids, primarily by modifying the formation of intermediates during heating in sulfuric acid (3). Addition of borate did not reduce interference from neutral sugars, however. Galambos (5) observed that sulfamate added before the first heating in sulfuric acid greatly lowered the browning due to neutral sugars and uranic acids, and borate increased the sensitivity of detection of certain uranic acids. High concentrations of sulfamate substantially suppressed color production of uranic acids with the carbazole. Knutsen and Jeanes (6) found that the carbazole analysis at 55°C rather than at 100°C reduced interference by neutral sugars without addition of sulfamate. The browning of neutral sugars during the first heating in sulfuric acid is substantial, but a vast majority of the color production from neutral sugars comes from the second heating after addition of carbazole. By far, the most widely used method today is that of Blumenkrantz and Asboe-Hansen (7), who introduced a new reagent, m-hydroxydiphenyl, to replace carbazole. After initial heating to hydrolyze polymers containing the uranic acids, reactions with m-hydroxydiphenyl are carried out at ambient temperature, thus avoiding the second heating that gives substantial interference by neutral sugars. They also found, however, that sulfamate precipitated upon cooling and was unsuitable in this reaction (7). From our experience, the m-hydroxydiphenyl reaction of Blumenkrantz and Asboe-Hansen (7) is quite suitable for pectins and polymers in which uranic acids compose a large amount of the sugar. When the uranic acids are a small proportion of the total sugar, however, nonspecific color produced by neutral sugars still poses a problem in the accurate determination of uranic acids, 157
Inc. reserved.
158
FILISETTI-COZZI
AND
CARPITA
TABLE1
Effect of Sulfamate on Interference
Without Sugars
sulfamate
Carbazole
2 pm01 Glc Gal Ara XYl 4 firno Glc Gal Ara XYl
Color Production by Neutral Sugars’
With
m-Hydroxydiphenyl
Carbazole
sulfamate m-Hydroxydiphenyl
1.352 0.881 0.315 0.424
0.557 0.505 0.148 0.095
0.129 0.171 0.057 0.024
0.053 0.057 0.061 0.000
>2.000 >2.000 0.524 0.838
1.277 1.215 0.238 0.181
0.258 0.458 0.114 0.043
0.152 0.253 0.072 0.024
a Sugars were dissolved in 0.4 ml of water, and 40 ~1 of 4 M sulfamic acid/potassium sulfamate, pH 1.6, was added to appropriate samples. After mixing, 2.4 ml of H,SOI (without borate) was added by repipet. After boiling and return to ambient temperature, 40 ~1 of 0.15% m-hydroxydiphenyl in 0.5% NaOH was overlaid in some samples and mixed in by vortex. In other samples, 100 ~1 of 0.1% carbazole in ethanol was overlaid and mixed into the samples, and the mixtures were boiled for 15 min for color production. All samples were read at 525 nm.
principally from the browning that occurs in the first heating. The addition of sulfamate substantially reduces this browning, but the reaction of neutral sugars and carbazole still produces some color in the second heating, and we have integrated from absorbance spectra the peak of the chromogen for a more reliable deter-
mination of uranic acid (8). We reevaluated the efficacy of combining these two well-known techniques and found that neutral sugar interference could be virtually eliminated by addition of small amounts of sulfamate and that uranic acids could still be measured with m-hydroxydiphenyl in the presence of sulfamate.
TABLE2
Influence of Sulfamate and Borate on Color Production by Uranic Acids, an Aminosugar, and Uranic Acid-Containing Polysaccharides”
Without Borate
Sample 200 nmol GalA
+ +
GlcA ManA
+ -c
GlcNAc 40 fig: PGA Alginic Gellan
+ acid
+ +
Carbazole
With
sulfamate m-Hydroxydiphenyl
Carbazole
sulfamate m-Hydroxydiphenyl
1.192 1.164 0.959 1.318 0.060 0.928 0.013 0.043
1.035 1.020 0.866 1.139 0.039 0.780 0.001 0.012
0.910 0.788 0.748 0.919 0.032 0.628 0.003 0.007
0.718 0.775 0.610 0.835 0.023 0.633 0.003 0.001
1.329 1.199 0.432 1.681 0.304 0.412
1.201 1.020 0.456 1.544 0.246 0.310
1.003 0.961 0.339 1.294 0.187 0.247
0.816 0.798 0.290 0.974 0.160 0.193
a Assays were carried out as outlined in Table 1 except that 80 ~1 of the m-hydroxydiphenyl reagent contained 75 mM sodium tetraborate. All samples were read at 525 nm.
reagent
was used and, where
noted,
the H,SO,
ASSAY
1.0
FOR
THE
MEASUREMENT
b. L
0.8 Ir, 2
a
0.6
0.4
,r \
0.2
0.0
\
- '\
0
l\
Gal
o-m-. 40
/ 80
.
.-
120
160
Volume of Sulfamate, pL
OF
URONIC
159
ACIDS
ml of concentrated H,SO, alone or containing up to 120 mM sodium tetraborate. In some experiments, up to 160 ~1 of 4 M sulfamic acid-potassium sulfamate (pH 1.6) was added to the solution of sugar and mixed thoroughly before addition of the sulfuric acid. The samples were heated to about 100°C in a boiling water bath for 20 min in 6 ml tubes capped with marbles to prevent condensation from contaminating the sample. The tubes were chilled in an ice bath, and 100 ~1 of the carbazole or up to 150 of ~1 of the m-hydroxydiphenyl reagent was added. The tubes with carbazole were heated to 100°C for 20 min and let cool to ambient temperature whereas the tubes with m-hydroxydiphenyl were incubated at ambient temperature for 15 min to 1 h before measurement of absorbance. Spectroscopy. Absorbances of reaction mixtures and appropriate blanks were either scanned from 400 to 700 nm (scanning rate, 750 nm/min) or recorded at 525 nm
FIG. 1. Suppression of color production by increasing amounts of the 4 M sulfamic acid-potassium sulfamate solution. The D-galaCturanic acid (200 nmol) and galactose (2 rmol) were dissolved in water, and up to 160 ~1 of the sulfamate reagent was added before addition of sulfuric acid (without borate). Eighty microliters of the m-hydroxydipbenyl reagent was used for development of color.
MATERIALS
AND
A
METHODS
Reagents. Sulfamic acid (Fisher Scientific) was stirred vigorously in one-half final volume of water, and saturated KOH was added dropwise until the sulfamic acid was dissolved. After the sulfamate solution had cooled, the pH was adjusted to 1.6, and the sulfamate was brought to 4 M with water and stored at ambient temperature. Analytical grade sulfuric acid (Mallinkrodt) was used in all experiments, and, when needed, solid sodium tetraborate (Pfaltz-Bauer) was dissolved to the desired concentration by stirring overnight in H,SO,. A 0.15% (w/v) solution of m-hydroxydiphenyl (Pfaltz-Bauer) in 0.5% (w/v) NaOH was prepared in advance and stored in a refrigerator in darkness. Prepared solutions of m-hydroxydiphenyl are stable for several weeks (7) but were used within 2 weeks in experiments described here. A 0.1% solution of carbazole (J. T. Baker) in absolute ethanol was freshly prepared. L-Arabinose, D-galactose, D-glucose, D-mannose, L-rhamnose, D-xylose, D-glucuronic acid, and D-galacturanic acid were from Sigma. Polygalacturonic acid was from Polysciences, Inc., and alginic acid was from Aldrich Chemical Co. Mannuronic acid lactone was a gift from Dr. J. N. BeMiller, and Pseudomonas gellan was gift from Dr. R. Chandrasekaran, both from the Whistler Center for Carbohydrate Research, Purdue University. Reactions. To 0.4 ml of uranic acid (5 to 200 nmol) or neutral sugar (50 nmol to 4 pmol) in water was added 2.4
400
460
520
580
640
)O
Wavelength, nm FIG. 2. Absorbance spectra of chromogens produced by neutral sugars and uranic acid with either carbazole (100 ~1) or m-hydroxydiphenyl (40 ~1) in the presence or absence of sulfamate. In samples with sulfamate, 40 pl of the reagent was mixed with 0.4 ml of the sugar sample before addition of 2.4 ml of sulfuric acid (without borate). (A) Reactions with 2 amol of D-galactose. (B) Reactions with 200 nmol of D-galacturonic acid in the presence of 1 pmol of D-gahCtOSe.
160
FILISETTI-COZZI
0.8 -
0.6 -
.
0.0 ' 0
40
gal
0
ara
0
glc
.
v
h
rhm
0
flK3”
.
GalA alone
80
120
Volume of m-Hydroxydiphenyl,
I 160
FL
FIG. 3. Influence of amount of m-hydroxydiphenyl reagent on color production with 200 nmol of n-galacturonic acid in the presence of 2 rmol of several neutral sugars. The sugars and uranic acid mixtures were dissolved in water, and 40 ~1 of the sulfamate reagent was added before addition of sulfuric acid (without borate).
with a Beckman Model DU-68 single-beam spectrophotometer. RESULTS
AND
DISCUSSION
The browning that occurred with 2 and 4 pmol of neutral sugars and carbazole resulted in substantial absorbance at 525 nm (Table 1). Use of m-hydroxydiphenyl significantly lowered this interference but did not eliminate it. Addition of sulfamate lowered the interference even further such that color production in carbazole was at least 3-fold lower than that produced with m-hydroxydiphenyl without sulfamate (Table 1). Color production by neutral sugars in reactions with m-hydroxydiphenyl plus sulfamate was at least 2-fold less than that in reactions with carbazole and sulfamate. The interference was essentially eliminated at amounts of sugar lo-fold higher than the amounts of uranic acid. Some sensitivity was compromised when m-hydroxydiphenyl was used rather than carbazole, but color production with authenic uranic acid was only about 15% less (Table 2). Sulfamate also lowered the color production by both carbazole and m-hydroxydiphenyl, but absorbances of the chromogens produced by D-galacturonic acid were still 68 and 76%, respectively, of reactions without sulfamate. Contrary to data of Blumenkrantz and Asboe-Hansen (7), carbazole produced more chromogen than did m-hydroxydiphenyl. N-Acetylglucosamine produced no chromagen with either carbazole or m-hydroxydiphenyl in the presence or absence of borate (Table 2).
AND
CARPITA
If even higher proportions of neutral sugar are present in samples containing uranic acid, for example, with 4 pmol of hexose, interference again becomes a problem. One potential solution might be to increase the amount of sulfamate. Maximal reduction of interference by neutral sugar was achieved with 40 ~1 of sulfamate, however, and additional sulfamate only lowered subsequent color production by uranic acid and the diphenyl reagent (Fig. 1). Precipitates formed only with 120 ~1 of 4 M sulfamate and higher, and they had to be removed by centrifugation before measurement. Although formation of some potassium sulfamate was necessary to prepare the 4 M solution, the final pH of the reagent was unimportant. We varied the pH of the 4 M sulfamic acid-potassium sulfamate from 1.6 to 4.9 with no effect on ultimate color production by 200 nmol of D-galacturanic acid (not shown). If sulfamate is used to reduce interference by neutral sugars, however, the volumes of the additions must be consistent for accurate measurement of uranic acid (Fig. 1). Not only was the color of neutral sugars markedly reduced by sulfamate but the production of nonspecific color from hexose by carbazole was also eliminated with m-hydroxydiphenyl (Figs. 2A and 2B). Another way to correct for interference by a great excess of neutral sugar is to scan the absorbance of the reaction mixtures from 400 to 700 nm and integrate the broad peak of absorbance due to uranic acid after subtraction of b; :kground between 430 and 650 nm (Ref. (8); Figs. 2A and 2B).
1.0 GlcA
o.o+ 0
40
80
Sodium Tetraborate,
120
mM
FIG. 4. Influence of sodium tetraborate on color production by several uranic acids. Forty microliters of the sulfamate reagent was added to each sample before addition of sulfuric acid. The borate was dissolved to the desired concentration in the sulfuric acid reagent, and 200 nmol of each uranic acid was detected with 80 pl of the m-hydroxydiphenyl reagent.
ASSAY
GalA+lOxGal,
FOR
THE
MEASUREMENT
no sulf ,/
t
0
40
80
Galacturonic
120
160
200
Acid, nmol
FIG.
5. Standard curve of D-galacturonic acid in the presence or absence of a lo-fold excess of D-galactose. D-Calacturonic acid (5 to 200 nmol) and D-gahCtOSe (50 to 2000 nmol) were dissolved in 0.4 ml of water. Sulfamate reagent (40 gl) was added to appropriate samples, and 2.4 ml of sulfuric acid with 75 mM sodium tetraborate was used. Color development was with 80 pl of the m-hydroxydiphenyl reagent.
Because the combination of sulfamate and m-hydroxydiphenyl markedly reduced the interference by neutral sugars, we were able to note that color production by uranic acid was actually less than expected in the presence of 1 pmol or more of some neutral sugars, particularly L-arabinose, D-maDDOSe, and L-rhamnose. Color production was enhanced to a certain extent by addition of more m-hydroxydiphenyl (Fig. 3). With Dgalacturonic acid alone, maximum color production was achieved with 40 to 60 ~1 of the reagent, but 80 ~1 of the reagent was needed to maximize color production by uranic acid in excess sugar. Addition of m-hydroxydiphenyl in excess of 80 ~1 reduced the color production (Fig. 3), mainly because of more rapid fading of the color upon standing. Addition of sodium tetraborate to the sulfuric acid proved essential for the detection of mannuronic acid lactone (Fig. 4). Sensitivity to D-glucuronic acid was increased by addition of small amounts of borate, but sensitivity to D-galacturonic acid was essentially unaffected. In samples that contain both D-mannuronic acid and D-glucuronic acid, we recommend that borate be included in the sulfuric acid reagent for accurate estimation of total uranic acid. With 40 ~1 of 4 M sulfamate and H,SO, containing 75 mM borate, color production by 80 ~1 of m-hydroxydiphenyl was linear from 5 to 200 nmol of uranic acid and nearly identical in the presence or absence of a lo-fold excess of D-galactose (Fig. 5). With sulfamate, m-hydroxydiphenyl, and sulfuric acid plus borate, plant polygalacturonic acid, algal al-
OF
URONIC
ACIDS
161
ginic acid, and Pseudomonas gellan produced chromagen close to expected values (Table 2). On the basis of a unit weight of 176.2 for D-galactosyluronic acid, 40 pg of PGA should yield 227 nmol of uranic acid. An absorbance of 0.798 yielded in the presence of borate corresponded to 206 nmol when compared to D-galacturonic acid standards (Table 2). Likewise, Pseudomonas gellan, a bacterial extracellular polysaccharide containing D-glucose, L-rhamnose, and D-glucuronic acid in a molar ratio of 2:l:l (9) gave close to stoichiometric values (Table 2). As expected, borate was essential for the determination of the D-mannuronic acid-rich alginic acid, a mixture of D-mannuronic acid and L-gulonic acid, but an absorbance of 0.974 corresponded to a substantial overestimate of 308 nmol when compared to mannuronic acid lactone standards (Table 2). We did not have the D-gulonic acid standards to make a more accurate determination. The alginate serves as an important example of the differences in color production that exist among the uranic acids, even with an optimized borate concentration in the sulfuric acid. Some attempts have been made to exploit differences in color production by various uranic acids in the presence and absence of borate and under varied reaction conditions (10). However, the advent of more reliable methods, particularly by HPLC and GLC-MS, has made possible more direct means for determining specific uranic acid constituents in complex mixtures. In many routine applications, such as in preliminary analyses and monitoring of column fractionations, the improved assay we suggest still provides a quick and reliable estimate of total uranic acid in the presence of excess neutral sugar. Recommended Procedures We propose that a combination of the methods of Galambos (5) and Blumenkrantz and Asboe-Hansen (7), i.e., a combination of sulfamate and m-hydroxydiphenyl, be incorporated in a routine assay of total uronic acids in the presence of excess neutral sugar. Samples containing up to 200 nmol of glycosyluronic acid are dissolved or suspended in 0.4 ml of water or dilute acetate buffer in small glass tubes, and 40 ~1 of 4 M sulfamic acid-potassium sulfamate (pH 1.6) is added and mixed thoroughly. Analytical grade (96.4% assay) H,SO, containing 75 IDM sodium tetraborate (2.4 ml) is then added, and the solution is stirred vigorously by vortex mixing. The solutions are heated to near 100°C for 20 min in a boiling water bath with the tubes capped with marbles. The tubes are placed in an ice bath to quickly cool the reaction mixtures to ambient temperature. After cooling, 80 ~1 of 0.15% (w/v) m-hydroxydiphenyl in 0.5% (w/v) NaOH is overlaid and then stirred in vigorously by vortex mixing. The pink color develops to completion in about 5 to 10 min and is stable for
162
FILISETTI-COZZI
about 1 h before fading results in some loss in color. Absorbance is read at 525 nm or, if browning is still observed, the reaction mixture can be scanned from 400 to 700 nm and the peak absorbance can be integrated after subtraction of background from 430 to 650 nm.
AND
REFERENCES 1. Dische, 2. Dische,
3. 4. 5. 6.
ACKNOWLEDGMENTS This work was supported by Development International BankUniversity of Sao Paul0 Project 09.05/161 and Contract DE-FG0288ER13903 from the Biological Energy Research, U.S. Department of Energy. This paper originally appeared as Journal Paper 12,890 of the Purdue University Agricultural Experiment Station. We thank Ms. Kimberly Jesionowsky and Michele Fagg for technical assistance.
CARPITA
7.
Z. (1947) Z. (1950)
J. Biol.
C&m.
167.189-198.
J. Biol. Chem. 183,489-494. Gregory, J. D. (1960) Arch. Biochem. Biophys. 89,X7-159. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4,330-334. Galambos, J. T. (1967) And. Biochem. 19, 119-132. Knutson, C. A., and Jeanes, A. (1968) Anal. Biochem. 24, 470481. Blumenkrantz, N., and Asboe-Hansen, G. (1973) Ad. Biochem. 54,484-489. Carpita, N. C. (1982) Phytochemistry 21, 1563-1566.
8. 9. O’Neill, M. A., Selvendran, hydr. Res. 124,123-133. 10. Knutson,
490.
C. A., and Jeanes,
R. R., and Morris, A. (1968)
V. J. (1983)
Carbo-
Anal. Biochem. 24, 482-
BIOCHEMISTRY
(1991)
197,157-162
Measurement of Uranic Acids without from Neutral Sugars Tullia
M. C. C. Filisetti-Cozzi*
and Nicholas
C. Carpita?,’
*Department0 de Alimentos e Nutri@o Experimental da Faculdade Postal 30786, CEP 01051, S6o Paul0 SP, Brazil; and TDepartment Purdue University, West Lafayette, Indiana 47907
Received
February
Carbazole was introduced for the quantitative determination of uranic acid in concentrated sulfuric acid (1,2). In the presence of other sugars, particularly hexoses, substantial interference that compromised specificity was observed, and the method was modified in
0003-2697/91 Copyright All rights
de Ci&cias Farmachticas, Universidade of Botany and Plant Pathology,
S&o Paulo,
27,199l
Replacement of carbazole with meta-hydroxydiphenyl greatly improves the determination of uranic acids in the presence of neutral sugars by preventing substantially, but not completely, the browning that occurs during the heating of sugars in concentrated sulfuric acid and avoiding the formation of additional interference by the carbazole reagent (Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54,484489). However, interference is still substantial when uranic acids are determined in the presence of excess neutral sugar, particularly because of the browning that occurs during the first heating before addition of the diphenyl reagent. The browning can be essentially eliminated by addition of sulfamate to the reaction mixture (Galambos, J. T. (1967) And. Biochem. 19, 119132). Although others have reported that sulfamate and the diphenyl reagent were incompatible, we find that a small amount of sulfamate suppresses color production by a 20-fold excess of some neutral sugars without substantial sacrifice of the sensitive detection of uranic acids by the diphenyl reagent. Sodium tetraborate is required for the detection of D-mannuronic acid and enhances color production by D-ghCUrOniC acid. We propose this modified sulfamatelm-hydroxydiphenyl assay as a rapid and reliable means for the assay of uranic acids, particularly when present in much smaller o 1991 Academic press, IIW. amOUntS than neutral sugars.
1 To whom
Interference
correspondence
should
$3.00 0 1991 by Academic Press, of reproduction in any form
be addressed.
several ways to reduce color production by neutral sugars. Gregory (3) and Bitter and Muir (4) observed that addition of borate to the carbazole reaction increased color production by certain uranic acids, primarily by modifying the formation of intermediates during heating in sulfuric acid (3). Addition of borate did not reduce interference from neutral sugars, however. Galambos (5) observed that sulfamate added before the first heating in sulfuric acid greatly lowered the browning due to neutral sugars and uranic acids, and borate increased the sensitivity of detection of certain uranic acids. High concentrations of sulfamate substantially suppressed color production of uranic acids with the carbazole. Knutsen and Jeanes (6) found that the carbazole analysis at 55°C rather than at 100°C reduced interference by neutral sugars without addition of sulfamate. The browning of neutral sugars during the first heating in sulfuric acid is substantial, but a vast majority of the color production from neutral sugars comes from the second heating after addition of carbazole. By far, the most widely used method today is that of Blumenkrantz and Asboe-Hansen (7), who introduced a new reagent, m-hydroxydiphenyl, to replace carbazole. After initial heating to hydrolyze polymers containing the uranic acids, reactions with m-hydroxydiphenyl are carried out at ambient temperature, thus avoiding the second heating that gives substantial interference by neutral sugars. They also found, however, that sulfamate precipitated upon cooling and was unsuitable in this reaction (7). From our experience, the m-hydroxydiphenyl reaction of Blumenkrantz and Asboe-Hansen (7) is quite suitable for pectins and polymers in which uranic acids compose a large amount of the sugar. When the uranic acids are a small proportion of the total sugar, however, nonspecific color produced by neutral sugars still poses a problem in the accurate determination of uranic acids, 157
Inc. reserved.
158
FILISETTI-COZZI
AND
CARPITA
TABLE1
Effect of Sulfamate on Interference
Without Sugars
sulfamate
Carbazole
2 pm01 Glc Gal Ara XYl 4 firno Glc Gal Ara XYl
Color Production by Neutral Sugars’
With
m-Hydroxydiphenyl
Carbazole
sulfamate m-Hydroxydiphenyl
1.352 0.881 0.315 0.424
0.557 0.505 0.148 0.095
0.129 0.171 0.057 0.024
0.053 0.057 0.061 0.000
>2.000 >2.000 0.524 0.838
1.277 1.215 0.238 0.181
0.258 0.458 0.114 0.043
0.152 0.253 0.072 0.024
a Sugars were dissolved in 0.4 ml of water, and 40 ~1 of 4 M sulfamic acid/potassium sulfamate, pH 1.6, was added to appropriate samples. After mixing, 2.4 ml of H,SOI (without borate) was added by repipet. After boiling and return to ambient temperature, 40 ~1 of 0.15% m-hydroxydiphenyl in 0.5% NaOH was overlaid in some samples and mixed in by vortex. In other samples, 100 ~1 of 0.1% carbazole in ethanol was overlaid and mixed into the samples, and the mixtures were boiled for 15 min for color production. All samples were read at 525 nm.
principally from the browning that occurs in the first heating. The addition of sulfamate substantially reduces this browning, but the reaction of neutral sugars and carbazole still produces some color in the second heating, and we have integrated from absorbance spectra the peak of the chromogen for a more reliable deter-
mination of uranic acid (8). We reevaluated the efficacy of combining these two well-known techniques and found that neutral sugar interference could be virtually eliminated by addition of small amounts of sulfamate and that uranic acids could still be measured with m-hydroxydiphenyl in the presence of sulfamate.
TABLE2
Influence of Sulfamate and Borate on Color Production by Uranic Acids, an Aminosugar, and Uranic Acid-Containing Polysaccharides”
Without Borate
Sample 200 nmol GalA
+ +
GlcA ManA
+ -c
GlcNAc 40 fig: PGA Alginic Gellan
+ acid
+ +
Carbazole
With
sulfamate m-Hydroxydiphenyl
Carbazole
sulfamate m-Hydroxydiphenyl
1.192 1.164 0.959 1.318 0.060 0.928 0.013 0.043
1.035 1.020 0.866 1.139 0.039 0.780 0.001 0.012
0.910 0.788 0.748 0.919 0.032 0.628 0.003 0.007
0.718 0.775 0.610 0.835 0.023 0.633 0.003 0.001
1.329 1.199 0.432 1.681 0.304 0.412
1.201 1.020 0.456 1.544 0.246 0.310
1.003 0.961 0.339 1.294 0.187 0.247
0.816 0.798 0.290 0.974 0.160 0.193
a Assays were carried out as outlined in Table 1 except that 80 ~1 of the m-hydroxydiphenyl reagent contained 75 mM sodium tetraborate. All samples were read at 525 nm.
reagent
was used and, where
noted,
the H,SO,
ASSAY
1.0
FOR
THE
MEASUREMENT
b. L
0.8 Ir, 2
a
0.6
0.4
,r \
0.2
0.0
\
- '\
0
l\
Gal
o-m-. 40
/ 80
.
.-
120
160
Volume of Sulfamate, pL
OF
URONIC
159
ACIDS
ml of concentrated H,SO, alone or containing up to 120 mM sodium tetraborate. In some experiments, up to 160 ~1 of 4 M sulfamic acid-potassium sulfamate (pH 1.6) was added to the solution of sugar and mixed thoroughly before addition of the sulfuric acid. The samples were heated to about 100°C in a boiling water bath for 20 min in 6 ml tubes capped with marbles to prevent condensation from contaminating the sample. The tubes were chilled in an ice bath, and 100 ~1 of the carbazole or up to 150 of ~1 of the m-hydroxydiphenyl reagent was added. The tubes with carbazole were heated to 100°C for 20 min and let cool to ambient temperature whereas the tubes with m-hydroxydiphenyl were incubated at ambient temperature for 15 min to 1 h before measurement of absorbance. Spectroscopy. Absorbances of reaction mixtures and appropriate blanks were either scanned from 400 to 700 nm (scanning rate, 750 nm/min) or recorded at 525 nm
FIG. 1. Suppression of color production by increasing amounts of the 4 M sulfamic acid-potassium sulfamate solution. The D-galaCturanic acid (200 nmol) and galactose (2 rmol) were dissolved in water, and up to 160 ~1 of the sulfamate reagent was added before addition of sulfuric acid (without borate). Eighty microliters of the m-hydroxydipbenyl reagent was used for development of color.
MATERIALS
AND
A
METHODS
Reagents. Sulfamic acid (Fisher Scientific) was stirred vigorously in one-half final volume of water, and saturated KOH was added dropwise until the sulfamic acid was dissolved. After the sulfamate solution had cooled, the pH was adjusted to 1.6, and the sulfamate was brought to 4 M with water and stored at ambient temperature. Analytical grade sulfuric acid (Mallinkrodt) was used in all experiments, and, when needed, solid sodium tetraborate (Pfaltz-Bauer) was dissolved to the desired concentration by stirring overnight in H,SO,. A 0.15% (w/v) solution of m-hydroxydiphenyl (Pfaltz-Bauer) in 0.5% (w/v) NaOH was prepared in advance and stored in a refrigerator in darkness. Prepared solutions of m-hydroxydiphenyl are stable for several weeks (7) but were used within 2 weeks in experiments described here. A 0.1% solution of carbazole (J. T. Baker) in absolute ethanol was freshly prepared. L-Arabinose, D-galactose, D-glucose, D-mannose, L-rhamnose, D-xylose, D-glucuronic acid, and D-galacturanic acid were from Sigma. Polygalacturonic acid was from Polysciences, Inc., and alginic acid was from Aldrich Chemical Co. Mannuronic acid lactone was a gift from Dr. J. N. BeMiller, and Pseudomonas gellan was gift from Dr. R. Chandrasekaran, both from the Whistler Center for Carbohydrate Research, Purdue University. Reactions. To 0.4 ml of uranic acid (5 to 200 nmol) or neutral sugar (50 nmol to 4 pmol) in water was added 2.4
400
460
520
580
640
)O
Wavelength, nm FIG. 2. Absorbance spectra of chromogens produced by neutral sugars and uranic acid with either carbazole (100 ~1) or m-hydroxydiphenyl (40 ~1) in the presence or absence of sulfamate. In samples with sulfamate, 40 pl of the reagent was mixed with 0.4 ml of the sugar sample before addition of 2.4 ml of sulfuric acid (without borate). (A) Reactions with 2 amol of D-galactose. (B) Reactions with 200 nmol of D-galacturonic acid in the presence of 1 pmol of D-gahCtOSe.
160
FILISETTI-COZZI
0.8 -
0.6 -
.
0.0 ' 0
40
gal
0
ara
0
glc
.
v
h
rhm
0
flK3”
.
GalA alone
80
120
Volume of m-Hydroxydiphenyl,
I 160
FL
FIG. 3. Influence of amount of m-hydroxydiphenyl reagent on color production with 200 nmol of n-galacturonic acid in the presence of 2 rmol of several neutral sugars. The sugars and uranic acid mixtures were dissolved in water, and 40 ~1 of the sulfamate reagent was added before addition of sulfuric acid (without borate).
with a Beckman Model DU-68 single-beam spectrophotometer. RESULTS
AND
DISCUSSION
The browning that occurred with 2 and 4 pmol of neutral sugars and carbazole resulted in substantial absorbance at 525 nm (Table 1). Use of m-hydroxydiphenyl significantly lowered this interference but did not eliminate it. Addition of sulfamate lowered the interference even further such that color production in carbazole was at least 3-fold lower than that produced with m-hydroxydiphenyl without sulfamate (Table 1). Color production by neutral sugars in reactions with m-hydroxydiphenyl plus sulfamate was at least 2-fold less than that in reactions with carbazole and sulfamate. The interference was essentially eliminated at amounts of sugar lo-fold higher than the amounts of uranic acid. Some sensitivity was compromised when m-hydroxydiphenyl was used rather than carbazole, but color production with authenic uranic acid was only about 15% less (Table 2). Sulfamate also lowered the color production by both carbazole and m-hydroxydiphenyl, but absorbances of the chromogens produced by D-galacturonic acid were still 68 and 76%, respectively, of reactions without sulfamate. Contrary to data of Blumenkrantz and Asboe-Hansen (7), carbazole produced more chromogen than did m-hydroxydiphenyl. N-Acetylglucosamine produced no chromagen with either carbazole or m-hydroxydiphenyl in the presence or absence of borate (Table 2).
AND
CARPITA
If even higher proportions of neutral sugar are present in samples containing uranic acid, for example, with 4 pmol of hexose, interference again becomes a problem. One potential solution might be to increase the amount of sulfamate. Maximal reduction of interference by neutral sugar was achieved with 40 ~1 of sulfamate, however, and additional sulfamate only lowered subsequent color production by uranic acid and the diphenyl reagent (Fig. 1). Precipitates formed only with 120 ~1 of 4 M sulfamate and higher, and they had to be removed by centrifugation before measurement. Although formation of some potassium sulfamate was necessary to prepare the 4 M solution, the final pH of the reagent was unimportant. We varied the pH of the 4 M sulfamic acid-potassium sulfamate from 1.6 to 4.9 with no effect on ultimate color production by 200 nmol of D-galacturanic acid (not shown). If sulfamate is used to reduce interference by neutral sugars, however, the volumes of the additions must be consistent for accurate measurement of uranic acid (Fig. 1). Not only was the color of neutral sugars markedly reduced by sulfamate but the production of nonspecific color from hexose by carbazole was also eliminated with m-hydroxydiphenyl (Figs. 2A and 2B). Another way to correct for interference by a great excess of neutral sugar is to scan the absorbance of the reaction mixtures from 400 to 700 nm and integrate the broad peak of absorbance due to uranic acid after subtraction of b; :kground between 430 and 650 nm (Ref. (8); Figs. 2A and 2B).
1.0 GlcA
o.o+ 0
40
80
Sodium Tetraborate,
120
mM
FIG. 4. Influence of sodium tetraborate on color production by several uranic acids. Forty microliters of the sulfamate reagent was added to each sample before addition of sulfuric acid. The borate was dissolved to the desired concentration in the sulfuric acid reagent, and 200 nmol of each uranic acid was detected with 80 pl of the m-hydroxydiphenyl reagent.
ASSAY
GalA+lOxGal,
FOR
THE
MEASUREMENT
no sulf ,/
t
0
40
80
Galacturonic
120
160
200
Acid, nmol
FIG.
5. Standard curve of D-galacturonic acid in the presence or absence of a lo-fold excess of D-galactose. D-Calacturonic acid (5 to 200 nmol) and D-gahCtOSe (50 to 2000 nmol) were dissolved in 0.4 ml of water. Sulfamate reagent (40 gl) was added to appropriate samples, and 2.4 ml of sulfuric acid with 75 mM sodium tetraborate was used. Color development was with 80 pl of the m-hydroxydiphenyl reagent.
Because the combination of sulfamate and m-hydroxydiphenyl markedly reduced the interference by neutral sugars, we were able to note that color production by uranic acid was actually less than expected in the presence of 1 pmol or more of some neutral sugars, particularly L-arabinose, D-maDDOSe, and L-rhamnose. Color production was enhanced to a certain extent by addition of more m-hydroxydiphenyl (Fig. 3). With Dgalacturonic acid alone, maximum color production was achieved with 40 to 60 ~1 of the reagent, but 80 ~1 of the reagent was needed to maximize color production by uranic acid in excess sugar. Addition of m-hydroxydiphenyl in excess of 80 ~1 reduced the color production (Fig. 3), mainly because of more rapid fading of the color upon standing. Addition of sodium tetraborate to the sulfuric acid proved essential for the detection of mannuronic acid lactone (Fig. 4). Sensitivity to D-glucuronic acid was increased by addition of small amounts of borate, but sensitivity to D-galacturonic acid was essentially unaffected. In samples that contain both D-mannuronic acid and D-glucuronic acid, we recommend that borate be included in the sulfuric acid reagent for accurate estimation of total uranic acid. With 40 ~1 of 4 M sulfamate and H,SO, containing 75 mM borate, color production by 80 ~1 of m-hydroxydiphenyl was linear from 5 to 200 nmol of uranic acid and nearly identical in the presence or absence of a lo-fold excess of D-galactose (Fig. 5). With sulfamate, m-hydroxydiphenyl, and sulfuric acid plus borate, plant polygalacturonic acid, algal al-
OF
URONIC
ACIDS
161
ginic acid, and Pseudomonas gellan produced chromagen close to expected values (Table 2). On the basis of a unit weight of 176.2 for D-galactosyluronic acid, 40 pg of PGA should yield 227 nmol of uranic acid. An absorbance of 0.798 yielded in the presence of borate corresponded to 206 nmol when compared to D-galacturonic acid standards (Table 2). Likewise, Pseudomonas gellan, a bacterial extracellular polysaccharide containing D-glucose, L-rhamnose, and D-glucuronic acid in a molar ratio of 2:l:l (9) gave close to stoichiometric values (Table 2). As expected, borate was essential for the determination of the D-mannuronic acid-rich alginic acid, a mixture of D-mannuronic acid and L-gulonic acid, but an absorbance of 0.974 corresponded to a substantial overestimate of 308 nmol when compared to mannuronic acid lactone standards (Table 2). We did not have the D-gulonic acid standards to make a more accurate determination. The alginate serves as an important example of the differences in color production that exist among the uranic acids, even with an optimized borate concentration in the sulfuric acid. Some attempts have been made to exploit differences in color production by various uranic acids in the presence and absence of borate and under varied reaction conditions (10). However, the advent of more reliable methods, particularly by HPLC and GLC-MS, has made possible more direct means for determining specific uranic acid constituents in complex mixtures. In many routine applications, such as in preliminary analyses and monitoring of column fractionations, the improved assay we suggest still provides a quick and reliable estimate of total uranic acid in the presence of excess neutral sugar. Recommended Procedures We propose that a combination of the methods of Galambos (5) and Blumenkrantz and Asboe-Hansen (7), i.e., a combination of sulfamate and m-hydroxydiphenyl, be incorporated in a routine assay of total uronic acids in the presence of excess neutral sugar. Samples containing up to 200 nmol of glycosyluronic acid are dissolved or suspended in 0.4 ml of water or dilute acetate buffer in small glass tubes, and 40 ~1 of 4 M sulfamic acid-potassium sulfamate (pH 1.6) is added and mixed thoroughly. Analytical grade (96.4% assay) H,SO, containing 75 IDM sodium tetraborate (2.4 ml) is then added, and the solution is stirred vigorously by vortex mixing. The solutions are heated to near 100°C for 20 min in a boiling water bath with the tubes capped with marbles. The tubes are placed in an ice bath to quickly cool the reaction mixtures to ambient temperature. After cooling, 80 ~1 of 0.15% (w/v) m-hydroxydiphenyl in 0.5% (w/v) NaOH is overlaid and then stirred in vigorously by vortex mixing. The pink color develops to completion in about 5 to 10 min and is stable for
162
FILISETTI-COZZI
about 1 h before fading results in some loss in color. Absorbance is read at 525 nm or, if browning is still observed, the reaction mixture can be scanned from 400 to 700 nm and the peak absorbance can be integrated after subtraction of background from 430 to 650 nm.
AND
REFERENCES 1. Dische, 2. Dische,
3. 4. 5. 6.
ACKNOWLEDGMENTS This work was supported by Development International BankUniversity of Sao Paul0 Project 09.05/161 and Contract DE-FG0288ER13903 from the Biological Energy Research, U.S. Department of Energy. This paper originally appeared as Journal Paper 12,890 of the Purdue University Agricultural Experiment Station. We thank Ms. Kimberly Jesionowsky and Michele Fagg for technical assistance.
CARPITA
7.
Z. (1947) Z. (1950)
J. Biol.
C&m.
167.189-198.
J. Biol. Chem. 183,489-494. Gregory, J. D. (1960) Arch. Biochem. Biophys. 89,X7-159. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4,330-334. Galambos, J. T. (1967) And. Biochem. 19, 119-132. Knutson, C. A., and Jeanes, A. (1968) Anal. Biochem. 24, 470481. Blumenkrantz, N., and Asboe-Hansen, G. (1973) Ad. Biochem. 54,484-489. Carpita, N. C. (1982) Phytochemistry 21, 1563-1566.
8. 9. O’Neill, M. A., Selvendran, hydr. Res. 124,123-133. 10. Knutson,
490.
C. A., and Jeanes,
R. R., and Morris, A. (1968)
V. J. (1983)
Carbo-
Anal. Biochem. 24, 482-
