ANALYTICAL
BIOCHEMISTRY
195,101-104
(1991)
Fluorometric Determination with 2-Aminothiophenol’
of Carbohydrate
Jian-Kang Zhu2 and Eugene A. Nothnagel Department of Botany and Plant Sciences,University of California, Riverside, California 92521
Received
January
11, 1991
The 2-aminothiophenol-based fluorometric assay of Nakano et cd. (1973, J. Pharm. Sot. Jpn. 93,360-353) for monosaccharides has been modified to improve the speed, applicability, and sensitivity of the method. The improved assay is applicable to complex carbohydrates as well as to monosaccharides. Less than 60 ngof carbohydrate in a final volume of 2 ml can be quantitatively measured within 30 min. The assay is reasonably compatible with the presence of a variety of reagents commonly used in aqueous buffer solutions. The assay is especially useful for monitoring column eluents during the purification of small quantities of carbohydrates or their conjugates. 0 1991 Academic Press, Inc.
In the course of our study of plant plasma membrane proteoglycans, we have developed a very sensitive fluorometric assay for the estimation of total carbohydrate. The sensitivity of this assay in a 2-ml final volume is better than 50 ng, and can be 10 ng for pentoses. Assays involving the use of phenol-sulfuric acid (l), anthrone (2), or orcinol(3) reagents are among the most commonly used for the estimation of carbohydrate. These calorimetric assays are simple and reproducible, but are sensitive only down to the microgram range. An extremely sensitive resorcinol-based fluorometric carbohydrate assay has been reported by Rogers et al. (4). In our hands, however, this assay does not yield reproducible results. The fluorometric assays of Lever (5), Honda et al, (6), and O’Neill et al. (7) are all applicable only to the quantitation of existing reducing ends of
i Supported by the National Science Foundation Cell Biology Program under Grant DCB-8716179. Partial support for purchase of the spectrofluorometer was provided by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health, under Grant BRSG SO7 RRO7010-19. ’ Present address: Department of Horticulture, Purdue University, West Lafayette, IN 47907. 0003-2697/91 Cr,nvr;mht
$3.00 cc=9 1441
sugars. The anthrone-based fluorometric assay for pentose developed by Hirayama et al. (8) is only slightly more sensitive than the calorimetric anthrone assay (2). The 2-aminothiophenol-based fluorometric assay of Nakano et al. (9) was developed for the determination of monosaccharides. In this assay, furfural and related molecules resulting from the dehydration of monosaccharides in strong acid are reacted with 2-aminothiophenol to form highly fluorescent 2-(2-furyl)benzothiazole and related fluorophores. As reported by Nakano et al. (9), the assay has relatively high sensitivity, but suffers from a long (3.5 h) reaction time in a boiling water bath and a large (20 ml) final volume. In this paper, we report on an improvement in the 2-aminothiophenolbased fluorometric assay. The modified assay is rapid, has high sensitivity, and is applicable to polysaccharides and glycoconjugates as well as to monosaccharides.
MATERIALS
AND
METHODS
2-Amirwthiophenol-HCl stock solutions. Reagent grade (99%) 2-aminothiophenol was purchased from Aldrich Chemical Co. (Milwaukee, WI). This reagent was routinely used as a saturated stock solution in dilute HCl. The saturated stock solution was prepared by adding 1.2 ml of 2-aminothiophenol to 98.8 ml of 120 mM HCl in water. The stock solution was stored in a dark bottle at room temperature for at least several days, and then aliquots were drawn from the supernatant for use in the assay. Stock solutions prepared and stored in this manner could be used to obtain consistent results in the assay for at least 2 months. As an alternative to the saturated stock solution, a 0.4% (w/v) solution of 2-aminothiophenol was prepared by mixing 0.342 ml of 2-aminothiophenol with 0.342 ml of ethanol, and then adding this mixture to a sufficient amount of 120 mM HCl in water to give 100 ml total volume. Use of this freshly prepared 0.4% stock solution in the assay gave results comparable to those obtained 101
h.,
Ar.Am..~r
P-n-e
T-r
102
ZHU
AND
when the saturated 2-aminothiophenol-HCl stock solution was used. Note that 2-aminothiophenol is a strongvesicant, and open solutions of this reagent should be handled with care in a fume hood. Carbohydrate stock solutions. Arabinose, xylose, ribose, fructose, glucose, galactose, mannose, rhamnose, glucuronic acid, galacturonic acid, glucosamine, sucrose, dextran (266 kDa), citrus pectin, and gum arabic were purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions were prepared by dissolving 1 mg of the desired carbohydrate in enough distilled water to give 100 ml total volume. H,SO, stock solution. Reagent grade (98%) H,SO, was purchased from Fisher Scientific (Pittsburgh, PA). A 30% (w/v) H,SO, stock solution was prepared by adding 16.6 ml of the reagent H,SO, to 80 ml of water, and then adding additional water to give 100 ml total volume. Assay procedure. The assay was carried out in 4-ml glass vials equipped with screw caps having Teflonfaced liners. To 0.5 ml of sample containing 0 to 1 pg of carbohydrate was added 0.2 ml of the 2-aminothiophenol-HCl stock solution (either saturated or 0.4%), followed by 0.5 ml of 30% (w/v) H,SO,. The vial was tightly capped, inverted several times for mixing, and then incubated in a dry-block heater thermostated at 150°C. After heating for 15 min, the vial was cooled to room temperature, and 0.8 ml of distilled water was added. The solution was transferred to a 1 X 1 X 4 cm glass cuvette, and fluorescence was measured using a Fluorolog 2 Model 112A spectrofluorometer (Spex Industries, Edison, NJ). The excitation spectrometer was set at 361 nm with 1.25 mm (4.5-nm bandpass) slits, and the double-emission spectrometer was set at 411 nm with 2.5 mm (4.5nm bandpass) slits. When spectral scans were recorded, the excitation spectra were corrected as described (lo), while the emission spectra were corrected by reference to the output of a standard lamp which was traceable to the National Bureau of Standards. RESULTS
In their work on the asOptimization of conditions. say of monosaccharides with 2-aminothiophenol, Nakano et al. (9) tested temperatures up to 100°C and found that the fluorescence intensity measured in the assay increased as the reaction temperature was increased. Even with the 3.5-h incubation time recommended by Nakano et al. (9), however, we found that relatively little fluorescence intensity resulted when complex carbohydrates such as gum arabic were reacted at 100°C. We arbitrarily selected 150°C for our assays, since at this temperature many complex carbohydrates can be readily hydrolyzed in moderate concentrations of
NOTHNAGEL
60
E
0
10 INCUBATION
20 TIME (min)
30
FIG. 1. Effect
of reaction time on the 2-aminothiophenol fluorometric assay. Assays with 1 pg of gum arabic (closed symbols) or a distilled water blank (open symbols) were carried out for various times at 150°C. Other conditions of the assay were as describedunder Materials and Methods. Data for gum arabic represent means + SD for three replicates. Data for the water blank represent means of two replicates.
H,SO,. At 15O”C, a 15-min incubation gave maximum fluorescence intensity with 1 pg of gum arabic as the sample (Fig. 1). Nakano et al. (9) reported that optimal fluorescence yield was obtained in the reaction at 100°C when the concentration of the H,SO, stock solution was 30% (w/v), which resulted in a H,SO, concentration of 7.5% (w/v) in the final assay mixture. We tested 30, 60, and 98% (w/v) concentrations of the H,SO, stock solution and found that the 30% concentration also gave maximum fluorescence in the assay reaction at 150°C (results not shown). The effect of the concentration of 2-aminothiophenol in the assay reaction at 150°C was also examined. The fluorescence intensity measured in the assay increased several fold as the concentration of the 2-aminothiophenol stock solution was increased to saturation from the 0.2% (w/v) level used by Nakano et al. (9) (results not shown). Thus, a saturated stock solution of 2-aminothiophenol was routinely used in the assay. Figure 2 shows the corrected excitation and emission spectra of the solution resulting from the assay of 500 ng of gum arabic under the conditions described under Materials and Methods. While Nakano et al. (9) used 365 and 430 nm as the excitation and emission wavelengths, respectively, the spectra of Fig. 2 show that the optimal excitation and emission wavelengths for the modified assay are 361 and 411 nm, respectively. These wavelengths were routinely used in the subsequent carbohydrate assays. Response with various carbohydrates. The relative responses measured in the fluorometric assays of various monosaccharides, oligosaccharides and polysaccharides are shown in Table 1. Pentoses gave the highest fluorescence intensities, while uranic acids gave relatively low intensities. Among all of the carbohydrates tested, glucosamine gave the lowest intensity.
FLUOROMETRIC
ASSAY
OF
-
3
)Q,
.,... _ .-....,..
_
Ara
_._
.
GA
--+--
Gal ’ Glc
-
0
100
E
Gum arabic is a commercially available arabinogalactan-protein that contains predominantly arabinosyl and galactosyl residues, lesser amounts of glucuronosyl and rhamnosyl residues, and about 2% protein (11). Consistent with this composition, the fluorescence intensity obtained with gum arabic was intermediate between the intensities obtained with arabinose and galactose (Table 1). Standard curves for the determination of galactose, glucose, xylose, arabinose and gum arabic are presented in Fig. 3A. The results show that the assay can easily detect 50 ng of any of these carbohydrates. The sensitiv-
Fluorometric Carbohydrate
Assay Responses Fluorescence
Note. triplicate, Methods. obtained
100.0 107.3 100.7 46.9 65.6 51.6 56.3 43.6 20.2 23.6 7.9 46.2 74.4 36.3 15.8
200
300
CARBOHYDRATE
3 iii
0
50
400
500
600
(ng)
200
ity is greatest for pentoses such as arabinose, where as little as 10 ng can be detected (Fig. 3B). Assessment of interference effects. The effects of various common laboratory reagents on the 2-aminothiophenol assay are shown in Table 2. Most of the reTABLE
2
Interference Effects of Various Reagents in the Fluorometric Assay
Carbohydrates Standard
150 (ng)
FIG. 3. (A) Standard curves for the 2-aminothiophenol fluorometric determination of xylose (Xyl), arabinose (Ara), gum arabic (GA), galactose (Gal) and glucose (Glc). Data represent means of three replicates. (B) Higher sensitivity range of the standard curve for the fluorometric determination of arabinose. Data represent means -t SD for three replicates. The assays were performed according to the procedure described under Materials and Methods.
1
of Various
100 ARABINOSE
deviation Added
Arabinose Xylose Ribose Glucose Galactose Mannose Rhamnose Fructose Glucuronic acid Galacturonic acid Glucosamine Sucrose Gum arabic Dextran Pectin
_._._
(nm)
FIG. 2. Corrected excitation and emission spectra of the solution resulting from the assay of 500 ng of gum arabic with 2-aminothiophenol, according to the procedure described under Materials and Methods. Emission at 470 nm was monitored for measurement of the excitation spectrum, while excitation was at 340 nm for measurement of the emission spectrum. The spectra shown are the results obtained after subtraction of the signals measured with the blank.
TABLE
.
520
420 WAVELENGTH
103
CARBOHYDRATE
4.5 2.5 0.9 0.3 0.1 1.8 0.6 2.3 2.4 0.4 0.4 1.7 3.9 2.6 1.1
600 ng of each of the following carbohydrates was assayed in according to the procedure described under Materials and Results shown are fluorescence intensities relative to that with arabinose.
reagent
Fluorescence
None 0.1 NH, acetate (pH 7.0) 0.1 Na acetate (pH 7.5) 0.1 imidazole-HCl (pH 7.5) 0.05 Tris-HCl (pH 7.0) 0.1 NaCl 0.5% (w/v) SDS” 0.5% (w/v) Triton X-100 0.1 mg/ml BSA 0.25 dithiothreitol M M M
M
M
mM
Note. Results shown are relative with 600 ng of arabinose assayed reagents (quadriplicates), according Materials and Methods. The final volume is indicated for each reagent. of the buffer before addition to the a SDS, sodium dodecyl sulfate. b BSA, bovine serum albumin.
100.0 115.8 111.3 113.2 123.7 118.5 147.9 137.4 120.7 101.6
Standard
deviation 4.5 3.1 1.3 5.5 2.7 2.5 2.7 1.3 4.4 1.7
fluorescence intensities measured in the presence of the indicated to the procedure described under concentration in the 2-ml assay Where indicated, the pH is that assay mixture.
104
ZHU
AND
NOTHNAGEL
ure 4 illustrates the use of the 2-aminothiophenol assay to monitor the elution of 150 pg of rose arabinogalactan-protein from a Sepharose CL-6B gel permeation column. DISCUSSION >wz i=
25
4E
0 30
40
50
FRACTION
60
70
80
NUMBER
FIG. 4. Application of the 2-aminothiophenol fluorometric assay to gel permeation chromatography of arabinogalactan-proteins from the plasma membrane of rose cells. Approximately 150 pg of arabinogalactan-protein was loaded on a Sepharose CL-6B column (100 cm x 0.88 cm’) and eluted with 50 mM ammonium acetate buffer (pH 6.9). Fractions of 1.3 ml were collected, and a 50-~1 aliquot was taken fram each fraction for carbohydrate determination according to the 2-aminothiophenol fluorometric procedure described under Materials and Methods.
agents had only modest effects on the fluorescence intensity. The reagents having the greatest effects were the detergents, which may act by altering the polarity of the environment of the fluorophores. The modest, positive nature of the interference effects observed with all of the reagents enables correction by use of appropriate blanks. Application of the assay. We have found this improved 2aminothiophenol assay to be very valuable in our study of arabinogalactan-proteins from the plasma membrane of “Paul’s Scarlet” rose (Rosa sp.) cells. Because very limited amounts of these macromolecules are available, it is not practical to use the phenol-sulfuric acid assay or other relatively insensitive assays to monitor elution profiles from chromatographic columns. Fig-
The fluorescence assay of Nakano et al. (9) has been modified to improve its sensitivity and reproducibility. The improved assay is also rapid, and relatively insensitive to interference from various reagents commonly used in biochemical buffer solutions. The assay should find many applications in carbohydrate research. ACKNOWLEDGMENT The authors ing the Nakano
thank Mr. Shigeru Tsukura for assistance in translatet al. (9) article from Japanese to English.
REFERENCES 1. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) And. Chem. 28,350-356. 2. Scott, T. A., and Melvin, E. H. (1953) Anal. Chem. 25.1656-1661. 3. Svennerholm, L. (1956) J. Neurochem. 1, 42-53. 4. Rogers, C. J., Chambers, Chem. 38,1851-1853.
C. W., and Clarke,
N. A. (1966)
Ad.
5. Lever, M. (1973) Biochem. Med. ‘7, 274-281. 6. Honda, S., Matsuda, Y., Takahashi, M., Kakehi, K., and Ganno, S. (1980) Anal. Chem. 52, 1079-1082. 7. O’Neill, R. A., Darvill, A., and Albersheim, P. (1989) Anal. Biohem. 177,11-15. H., Hiraki, K., and Nishikawa, Y. (1971) Jpn. Analyst 8. Hirayama, 20,1435-1441. [In Japanese] 9. Nakano, S., Taniguchi, (1973) J. Pharm. Sot. 10. Nothnagel, 11. Akiyama, 235-237.
H., Furuhashi,
Jpn. 93,350-353.
T., and Mikoshiba, [In Japanese]
E. A. (1987) And. Biochem. 163,224-237. Y., Eda, S., and Kato, L. (1984) Agric. Biol.
Chem.
K.
48,
BIOCHEMISTRY
195,101-104
(1991)
Fluorometric Determination with 2-Aminothiophenol’
of Carbohydrate
Jian-Kang Zhu2 and Eugene A. Nothnagel Department of Botany and Plant Sciences,University of California, Riverside, California 92521
Received
January
11, 1991
The 2-aminothiophenol-based fluorometric assay of Nakano et cd. (1973, J. Pharm. Sot. Jpn. 93,360-353) for monosaccharides has been modified to improve the speed, applicability, and sensitivity of the method. The improved assay is applicable to complex carbohydrates as well as to monosaccharides. Less than 60 ngof carbohydrate in a final volume of 2 ml can be quantitatively measured within 30 min. The assay is reasonably compatible with the presence of a variety of reagents commonly used in aqueous buffer solutions. The assay is especially useful for monitoring column eluents during the purification of small quantities of carbohydrates or their conjugates. 0 1991 Academic Press, Inc.
In the course of our study of plant plasma membrane proteoglycans, we have developed a very sensitive fluorometric assay for the estimation of total carbohydrate. The sensitivity of this assay in a 2-ml final volume is better than 50 ng, and can be 10 ng for pentoses. Assays involving the use of phenol-sulfuric acid (l), anthrone (2), or orcinol(3) reagents are among the most commonly used for the estimation of carbohydrate. These calorimetric assays are simple and reproducible, but are sensitive only down to the microgram range. An extremely sensitive resorcinol-based fluorometric carbohydrate assay has been reported by Rogers et al. (4). In our hands, however, this assay does not yield reproducible results. The fluorometric assays of Lever (5), Honda et al, (6), and O’Neill et al. (7) are all applicable only to the quantitation of existing reducing ends of
i Supported by the National Science Foundation Cell Biology Program under Grant DCB-8716179. Partial support for purchase of the spectrofluorometer was provided by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health, under Grant BRSG SO7 RRO7010-19. ’ Present address: Department of Horticulture, Purdue University, West Lafayette, IN 47907. 0003-2697/91 Cr,nvr;mht
$3.00 cc=9 1441
sugars. The anthrone-based fluorometric assay for pentose developed by Hirayama et al. (8) is only slightly more sensitive than the calorimetric anthrone assay (2). The 2-aminothiophenol-based fluorometric assay of Nakano et al. (9) was developed for the determination of monosaccharides. In this assay, furfural and related molecules resulting from the dehydration of monosaccharides in strong acid are reacted with 2-aminothiophenol to form highly fluorescent 2-(2-furyl)benzothiazole and related fluorophores. As reported by Nakano et al. (9), the assay has relatively high sensitivity, but suffers from a long (3.5 h) reaction time in a boiling water bath and a large (20 ml) final volume. In this paper, we report on an improvement in the 2-aminothiophenolbased fluorometric assay. The modified assay is rapid, has high sensitivity, and is applicable to polysaccharides and glycoconjugates as well as to monosaccharides.
MATERIALS
AND
METHODS
2-Amirwthiophenol-HCl stock solutions. Reagent grade (99%) 2-aminothiophenol was purchased from Aldrich Chemical Co. (Milwaukee, WI). This reagent was routinely used as a saturated stock solution in dilute HCl. The saturated stock solution was prepared by adding 1.2 ml of 2-aminothiophenol to 98.8 ml of 120 mM HCl in water. The stock solution was stored in a dark bottle at room temperature for at least several days, and then aliquots were drawn from the supernatant for use in the assay. Stock solutions prepared and stored in this manner could be used to obtain consistent results in the assay for at least 2 months. As an alternative to the saturated stock solution, a 0.4% (w/v) solution of 2-aminothiophenol was prepared by mixing 0.342 ml of 2-aminothiophenol with 0.342 ml of ethanol, and then adding this mixture to a sufficient amount of 120 mM HCl in water to give 100 ml total volume. Use of this freshly prepared 0.4% stock solution in the assay gave results comparable to those obtained 101
h.,
Ar.Am..~r
P-n-e
T-r
102
ZHU
AND
when the saturated 2-aminothiophenol-HCl stock solution was used. Note that 2-aminothiophenol is a strongvesicant, and open solutions of this reagent should be handled with care in a fume hood. Carbohydrate stock solutions. Arabinose, xylose, ribose, fructose, glucose, galactose, mannose, rhamnose, glucuronic acid, galacturonic acid, glucosamine, sucrose, dextran (266 kDa), citrus pectin, and gum arabic were purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions were prepared by dissolving 1 mg of the desired carbohydrate in enough distilled water to give 100 ml total volume. H,SO, stock solution. Reagent grade (98%) H,SO, was purchased from Fisher Scientific (Pittsburgh, PA). A 30% (w/v) H,SO, stock solution was prepared by adding 16.6 ml of the reagent H,SO, to 80 ml of water, and then adding additional water to give 100 ml total volume. Assay procedure. The assay was carried out in 4-ml glass vials equipped with screw caps having Teflonfaced liners. To 0.5 ml of sample containing 0 to 1 pg of carbohydrate was added 0.2 ml of the 2-aminothiophenol-HCl stock solution (either saturated or 0.4%), followed by 0.5 ml of 30% (w/v) H,SO,. The vial was tightly capped, inverted several times for mixing, and then incubated in a dry-block heater thermostated at 150°C. After heating for 15 min, the vial was cooled to room temperature, and 0.8 ml of distilled water was added. The solution was transferred to a 1 X 1 X 4 cm glass cuvette, and fluorescence was measured using a Fluorolog 2 Model 112A spectrofluorometer (Spex Industries, Edison, NJ). The excitation spectrometer was set at 361 nm with 1.25 mm (4.5-nm bandpass) slits, and the double-emission spectrometer was set at 411 nm with 2.5 mm (4.5nm bandpass) slits. When spectral scans were recorded, the excitation spectra were corrected as described (lo), while the emission spectra were corrected by reference to the output of a standard lamp which was traceable to the National Bureau of Standards. RESULTS
In their work on the asOptimization of conditions. say of monosaccharides with 2-aminothiophenol, Nakano et al. (9) tested temperatures up to 100°C and found that the fluorescence intensity measured in the assay increased as the reaction temperature was increased. Even with the 3.5-h incubation time recommended by Nakano et al. (9), however, we found that relatively little fluorescence intensity resulted when complex carbohydrates such as gum arabic were reacted at 100°C. We arbitrarily selected 150°C for our assays, since at this temperature many complex carbohydrates can be readily hydrolyzed in moderate concentrations of
NOTHNAGEL
60
E
0
10 INCUBATION
20 TIME (min)
30
FIG. 1. Effect
of reaction time on the 2-aminothiophenol fluorometric assay. Assays with 1 pg of gum arabic (closed symbols) or a distilled water blank (open symbols) were carried out for various times at 150°C. Other conditions of the assay were as describedunder Materials and Methods. Data for gum arabic represent means + SD for three replicates. Data for the water blank represent means of two replicates.
H,SO,. At 15O”C, a 15-min incubation gave maximum fluorescence intensity with 1 pg of gum arabic as the sample (Fig. 1). Nakano et al. (9) reported that optimal fluorescence yield was obtained in the reaction at 100°C when the concentration of the H,SO, stock solution was 30% (w/v), which resulted in a H,SO, concentration of 7.5% (w/v) in the final assay mixture. We tested 30, 60, and 98% (w/v) concentrations of the H,SO, stock solution and found that the 30% concentration also gave maximum fluorescence in the assay reaction at 150°C (results not shown). The effect of the concentration of 2-aminothiophenol in the assay reaction at 150°C was also examined. The fluorescence intensity measured in the assay increased several fold as the concentration of the 2-aminothiophenol stock solution was increased to saturation from the 0.2% (w/v) level used by Nakano et al. (9) (results not shown). Thus, a saturated stock solution of 2-aminothiophenol was routinely used in the assay. Figure 2 shows the corrected excitation and emission spectra of the solution resulting from the assay of 500 ng of gum arabic under the conditions described under Materials and Methods. While Nakano et al. (9) used 365 and 430 nm as the excitation and emission wavelengths, respectively, the spectra of Fig. 2 show that the optimal excitation and emission wavelengths for the modified assay are 361 and 411 nm, respectively. These wavelengths were routinely used in the subsequent carbohydrate assays. Response with various carbohydrates. The relative responses measured in the fluorometric assays of various monosaccharides, oligosaccharides and polysaccharides are shown in Table 1. Pentoses gave the highest fluorescence intensities, while uranic acids gave relatively low intensities. Among all of the carbohydrates tested, glucosamine gave the lowest intensity.
FLUOROMETRIC
ASSAY
OF
-
3
)Q,
.,... _ .-....,..
_
Ara
_._
.
GA
--+--
Gal ’ Glc
-
0
100
E
Gum arabic is a commercially available arabinogalactan-protein that contains predominantly arabinosyl and galactosyl residues, lesser amounts of glucuronosyl and rhamnosyl residues, and about 2% protein (11). Consistent with this composition, the fluorescence intensity obtained with gum arabic was intermediate between the intensities obtained with arabinose and galactose (Table 1). Standard curves for the determination of galactose, glucose, xylose, arabinose and gum arabic are presented in Fig. 3A. The results show that the assay can easily detect 50 ng of any of these carbohydrates. The sensitiv-
Fluorometric Carbohydrate
Assay Responses Fluorescence
Note. triplicate, Methods. obtained
100.0 107.3 100.7 46.9 65.6 51.6 56.3 43.6 20.2 23.6 7.9 46.2 74.4 36.3 15.8
200
300
CARBOHYDRATE
3 iii
0
50
400
500
600
(ng)
200
ity is greatest for pentoses such as arabinose, where as little as 10 ng can be detected (Fig. 3B). Assessment of interference effects. The effects of various common laboratory reagents on the 2-aminothiophenol assay are shown in Table 2. Most of the reTABLE
2
Interference Effects of Various Reagents in the Fluorometric Assay
Carbohydrates Standard
150 (ng)
FIG. 3. (A) Standard curves for the 2-aminothiophenol fluorometric determination of xylose (Xyl), arabinose (Ara), gum arabic (GA), galactose (Gal) and glucose (Glc). Data represent means of three replicates. (B) Higher sensitivity range of the standard curve for the fluorometric determination of arabinose. Data represent means -t SD for three replicates. The assays were performed according to the procedure described under Materials and Methods.
1
of Various
100 ARABINOSE
deviation Added
Arabinose Xylose Ribose Glucose Galactose Mannose Rhamnose Fructose Glucuronic acid Galacturonic acid Glucosamine Sucrose Gum arabic Dextran Pectin
_._._
(nm)
FIG. 2. Corrected excitation and emission spectra of the solution resulting from the assay of 500 ng of gum arabic with 2-aminothiophenol, according to the procedure described under Materials and Methods. Emission at 470 nm was monitored for measurement of the excitation spectrum, while excitation was at 340 nm for measurement of the emission spectrum. The spectra shown are the results obtained after subtraction of the signals measured with the blank.
TABLE
.
520
420 WAVELENGTH
103
CARBOHYDRATE
4.5 2.5 0.9 0.3 0.1 1.8 0.6 2.3 2.4 0.4 0.4 1.7 3.9 2.6 1.1
600 ng of each of the following carbohydrates was assayed in according to the procedure described under Materials and Results shown are fluorescence intensities relative to that with arabinose.
reagent
Fluorescence
None 0.1 NH, acetate (pH 7.0) 0.1 Na acetate (pH 7.5) 0.1 imidazole-HCl (pH 7.5) 0.05 Tris-HCl (pH 7.0) 0.1 NaCl 0.5% (w/v) SDS” 0.5% (w/v) Triton X-100 0.1 mg/ml BSA 0.25 dithiothreitol M M M
M
M
mM
Note. Results shown are relative with 600 ng of arabinose assayed reagents (quadriplicates), according Materials and Methods. The final volume is indicated for each reagent. of the buffer before addition to the a SDS, sodium dodecyl sulfate. b BSA, bovine serum albumin.
100.0 115.8 111.3 113.2 123.7 118.5 147.9 137.4 120.7 101.6
Standard
deviation 4.5 3.1 1.3 5.5 2.7 2.5 2.7 1.3 4.4 1.7
fluorescence intensities measured in the presence of the indicated to the procedure described under concentration in the 2-ml assay Where indicated, the pH is that assay mixture.
104
ZHU
AND
NOTHNAGEL
ure 4 illustrates the use of the 2-aminothiophenol assay to monitor the elution of 150 pg of rose arabinogalactan-protein from a Sepharose CL-6B gel permeation column. DISCUSSION >wz i=
25
4E
0 30
40
50
FRACTION
60
70
80
NUMBER
FIG. 4. Application of the 2-aminothiophenol fluorometric assay to gel permeation chromatography of arabinogalactan-proteins from the plasma membrane of rose cells. Approximately 150 pg of arabinogalactan-protein was loaded on a Sepharose CL-6B column (100 cm x 0.88 cm’) and eluted with 50 mM ammonium acetate buffer (pH 6.9). Fractions of 1.3 ml were collected, and a 50-~1 aliquot was taken fram each fraction for carbohydrate determination according to the 2-aminothiophenol fluorometric procedure described under Materials and Methods.
agents had only modest effects on the fluorescence intensity. The reagents having the greatest effects were the detergents, which may act by altering the polarity of the environment of the fluorophores. The modest, positive nature of the interference effects observed with all of the reagents enables correction by use of appropriate blanks. Application of the assay. We have found this improved 2aminothiophenol assay to be very valuable in our study of arabinogalactan-proteins from the plasma membrane of “Paul’s Scarlet” rose (Rosa sp.) cells. Because very limited amounts of these macromolecules are available, it is not practical to use the phenol-sulfuric acid assay or other relatively insensitive assays to monitor elution profiles from chromatographic columns. Fig-
The fluorescence assay of Nakano et al. (9) has been modified to improve its sensitivity and reproducibility. The improved assay is also rapid, and relatively insensitive to interference from various reagents commonly used in biochemical buffer solutions. The assay should find many applications in carbohydrate research. ACKNOWLEDGMENT The authors ing the Nakano
thank Mr. Shigeru Tsukura for assistance in translatet al. (9) article from Japanese to English.
REFERENCES 1. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) And. Chem. 28,350-356. 2. Scott, T. A., and Melvin, E. H. (1953) Anal. Chem. 25.1656-1661. 3. Svennerholm, L. (1956) J. Neurochem. 1, 42-53. 4. Rogers, C. J., Chambers, Chem. 38,1851-1853.
C. W., and Clarke,
N. A. (1966)
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