Clrirrlicw
cl IItJ/J’Ii(W
‘t-z,Elscvicr
SELECTIVE ACIDS BY SULFATE
SUSUMU TAKIURA
/dCItJ. 77 ( 1975) 199-203
Scientific
Publishing
Company,
199 Amstcrdnm
- Printed
in The Ncthcrluncls
MICRO DETERMINATION OF UNCONJUGATED URONIC FLUORESCENCE REACTIONS WITH ETHYLENEDIAMINE
HONDA.
KYOKO
SUDO.
KAZUAKI
KAKEHI.
HIDETAKA
YUKI
and
‘KIYOSHI
Recently, it has been shown that reactions of reducing sugars with cthylenediamine sulfate in phosphate buffer produce a fluorescence having excitation and emission maxima at 400 and 465 nm. respectively: and these reactions were successfully applied to the micro determination of reducing sugars’. Further studies on the reactions of a variety of carbohydrates with this amine salt under various conditions showed that. when acctnte buffer was used. another lluorcscence having excitaiion and emission maxima nt 328 and 406 nm. rcspectivcly. was formed specifically with uranic acids. This paper deals with a spectrofluorimetric method for the micro determination of uranic acids based on the latter reaction conditions. Under the neutral conditions employed, glycosidic linkages in aliphatic and aromatic uronides are not cleaved by hydrolysis. Therefore. unconjugated uranic acids can be determined nccurately without intcrfcrcnce from uronides.
Among
several
kinds
of buffer
solutions
I
I .Y
I
5
II
Fig. I. pH Dcpcndcncc of lluorcsccncc 2.OO*IO-J M: reaction time. 3 II. Fig. 2. Elkct reaction time.
having
of bulk 3 Ii.
concentration.
D-golacturonic
I’,
in 0.1
acid.
pH values.
I
,111
formalion
various
M
I
2
;ICC~;IIC
2.00~ IO-”
II.
,‘.I,
bufkr.
kf;
acetate
I
I 0..
I.
D-pulxturonic
LICCtiIIC bulkr.
11
wzid.
ptl
6.00:
S. HONDA,
200
K. SUDO.
K. KAKEI-11.
H. YUKI.
K. TAKIURA
buffer was found to give the most intense fluorescence at 328 (excitation)/405 nm (emission). In phosphate buffer another fluorescence was observed at 400/465 nm. as reported’. Figure 1 shows the pH dependence of fluorescence formation. The higher the pH of acetate buffer. the more intense the lluorescence:However. since very high pH values weaken the buffer effect, pH 6 was considered to be appropriate. Figure 2 shows the relationship between fluorescence intensity and the concentration of acetate buffer. The hyperbola indicates that fluorescence intensity decreased with increasing buffer concentrations. Therefore, a buffer concentration of 0.1 M was adopted in the standard procedure. Fluorescence formation was also dependent on the amount of the reagent if less than a IO-fold molar amount relative to uranic acids was present. but the use of larger excesses of the reagent gave a constant intensity of fluorescence. In the standard procedure the maximum concentration (2~ IO-’ M) of the reagent is used that allows a buffer effect, Heating almost
time The fluorescence intensity increased gradually for 3 h. Thereafter it. became constant. Accordingly. a heating time of 3 h was considered to be appropriate.
Fluorcscertce spectra und calibration The fluorescence spectrum for D-galacturonic acid obtained by the standard procedure is depicted in Fig. 3. There are observed excitation and emission maxima at 328 and 405 nm. respectively. D-Glucuronic acid as well as its &tone gave similar spectra. Calibration curves for D-galacturonic acid and D-glucuronic acid are shown in Fig. 4. The lines form gently sloping sigmoid curves which show linearity for sample concentrations of 5.0*10-“-1.5. IO-’ M and 2.0. lO-s-2.O. 10m4 M in both cases. The sensitivity of D-glucuronic acid was 82% lower than that of D-galacturonic acid. The low sensitivity is attributable to acid-&tone equilibration, since D-glucuronolactone gave the same intensity as D-glucuronic acid. D-Galacturonic acid does not equilibrate because it lacks the structural requirement' for
1.0 2. ” ._
‘2 0 ; ._
2 .” ._I
x
2
II.5
-
-11 SUU
Fig. 3. Fluoresccncc reaction time. 3 h.
4
spcclrum.
uu
D-pnlucturonic
1,111
ucid.
2.00. low4
M:
0.1 M ucctatc
buffer,
pH
G.00:
FLUORIMETRY
OF UNCONJUGATED
0,s
UROl’jIC
1.0
Fig. 4. Culibrution curves of uranic acids obtninod acid: (b) D-giucuronic ncid or D-lillcllronolactonc. TABLE
ACIDS
1.s
201
x
10.5
bl
by the stnndtird proccciurc.
(n) D-~iii~tcttlr~nic
I
REPRODUClBILlTY (5 dctcrmirwtions
OF DETERMINATION were done at cnch level.)
lactonization. Precision data for various concentrations of sample solutions are given in Table 1. The results indicate that this method is accurate and reproducible. EXPERIMENTAL
All reagents and samples of uranic acids were of reagent grade. Samples of uronides were kindly donated by Dr. K. Okui of Central Research Laboratories, Chugai Pll~~rm~~ceuticals Co.. Ltd. Fluorescence was measured with a Hitachi MPF-2A spectrofluorimetcr in l-cm quartz cells. To a buffered reagent solution (3.00 ml), prepared by mixing 2. lOs2 M ethylenediamine sulfate (I volume) and 0.1 M acetate buffer (pH 6.00, 2 volumes), add a sample solution (2.00 ml) containing 1.O* IO- z-3.0* IO- ’ or 4.0. IO- ‘-4.0 *IO- ’ llmofe of a uranic acid. Heat the mixture for 3 h on a boiling water bath. Determine the concentration of uranic acid by reading the fluorescence intensity at 328 (excitation) and 405 nm (emission) within 24 h. Prepare a calibration curve
S. HONDA,
202 simultaneously. distilled water. RESULTS
AND
A reagent
blank
is made
K. SUDO.
by
K. KAKEHI.
replacing
the
I-I. YUKI.
sample
K.TAKlURA
solution
with
DISCUSSION
Comparative studies under the standard reaction conditions indicated that these fluorescence reactions are highly selective for uranic acids. Various types of organic compounds cxamincd, including alcohols. phenols. umines. nitro compounds. kctoncs. carboxylic acids. esters. amides. nitrilcs, and sulfonic acids gave no fluorescence with ethylenediaminc sulfate. Some kinds of aromatic aldchydes, such as anisaldehyde and o-nitrobenzaldehyde. were the only exceptions. The relative fluorescence intensities at 328/405 nm. as referred to D-galacturonic acid, of all the 0.01 : aldoscs (glycol carbohydrates ( 1.O s IO- s M) listed below did not exceed aldehyde. DL-glyceraldehyde. D-erythrose. D-arab.inose. D-ribose. D-xylose. Dgalactose. D-glucose, D-mannose); ketoscs (D-fructose. L-sorbose); 2-deoxy sugars (2-deoxy-D-ribosc. 2-deoxy-D-glucose); methyl pcntoses (L-fucose. L-rhamnose); alditols (erythritol. D-xylitol. D-galactitol. D-mannitol. D-sorbitol): amino sugars (D-galactosamine hydrochloride. D-glucosamine hydrochloride. N-acetyl-D-galactosamine. N,-acetyl-D-glucosamine): aldonic acids (D-arabonic acid. D-gluconic acid); glycosides (methyl z- and /I-D-glucopyranosides. phenyl Q- and /3-D-glycopyranosides): oligosaccharides (maltose. cellobiose. lactose, sucrose, raffinose): polysacchitridcs (dcxtran. glycogcn. starch. chondroitin sulfates A and C). Determination of 1.0, lo-& M D-glucuronic acid was not affected by lo-fold molar amounts of D-glucose. but the prescncc of SL IOO-fold molar amount D-glucose caused considerable depression of fluorescence intensity for unknown reasons.
It is noticeable that. unlike acidic conditions, this fiuorimetric acids without cleaving glycosidic Accordingly, unconjugated uranic the presence of large amounts of TABLE
the calorimetric methodr-4 which USC strongly method makes it possible to determine uranic linkages in aliphatic and aromatic uronides. acids can bc determined without intcrfcrence in uronidcs. Uronides inspected include ethyl. n-
II
DETERMINATION
OF
D-GLUCURONIC
(In cuch cusc 2.00* lO-1 pmolc
D-.gIucumnic
ACID
IN THE
PRESENCE
OF D-GLUCURONIDES
acid wus used.)
Ethyl D-glucuronidc added (pmolc) * D-Glucuronic ocid found (pmolc~ 10bl)
0.20
0.40
0.60
0.80
I .M
I .2O
1.40
I.60
I.80
2.00
I .95
l.9H
2.0 I
I .9x
I .96
2.06
2.01
2.06
2.15
2.27
p-Tolyl D-glucuronidc uddcd (pmolc) D-Glucuronic acid round (pmolc. 10w2) --. -.___
0.20
0.40
0.60
0.x0
I.00
I .20
I .40
I .60
I.XO
2.00
I -94
I.96
I.98
I .97
I.98
‘*OS
2.07
2.06
2.18
2.25
_--.----
~--._.--_--.--
FLUORIMETRY
OF
UNCONJUGATED
URONIC
ACIDS
203
propyl. isobutyl. phenyl. p-tolyl. and p-chlorophcnyl glucuronides. Table II gives typical data obtnincd from ethyl and p-tolyl glucuronides. It is clear that 1.0. lo-” M D-glucuronic acid can be determined accurately in the presence of at least 80-fold molar amounts of D-glucuronides. The authors express their sincere gratitude to Dr. K. Okui for the gift of uronidc samples. The authors are also grateful to Chugai Pharmaceuticals Co.. Ltd. for financial support. SUMMARY
A simple method for the spectrofluorimetric determination of uranic acids is proposed. The fluorescence at 328 (cxcitution) and 405 nm (emission) formed by heating sumplc solutions in acetate buffer containing large amounts of cthylcncdiamine sulfntc. can be used to detcrminc 1.0. IO-“-3.0. 10m2 or 4.0. 10S2-4.0, lo- ’ /lmole of unconjugated uronic acids accurately. without intcrferencc from other carbohydrate materials. especially uronidcs. REFERENCES I S. Honcla. K. Kakimolo. K. Kakchi and K. Takiutx .‘lrlct/. Clrirtr. rlc./c/. 70 ( 1974) 133. 2 Z. Dischc. J. 13id. Chw.. I67 ( 1947) 180: .I. Rid. Clrotr.. 17 1 ( 1947) 725: J. Rio/. ( 1950) 4x9.
Chw..
183
cl IItJ/J’Ii(W
‘t-z,Elscvicr
SELECTIVE ACIDS BY SULFATE
SUSUMU TAKIURA
/dCItJ. 77 ( 1975) 199-203
Scientific
Publishing
Company,
199 Amstcrdnm
- Printed
in The Ncthcrluncls
MICRO DETERMINATION OF UNCONJUGATED URONIC FLUORESCENCE REACTIONS WITH ETHYLENEDIAMINE
HONDA.
KYOKO
SUDO.
KAZUAKI
KAKEHI.
HIDETAKA
YUKI
and
‘KIYOSHI
Recently, it has been shown that reactions of reducing sugars with cthylenediamine sulfate in phosphate buffer produce a fluorescence having excitation and emission maxima at 400 and 465 nm. respectively: and these reactions were successfully applied to the micro determination of reducing sugars’. Further studies on the reactions of a variety of carbohydrates with this amine salt under various conditions showed that. when acctnte buffer was used. another lluorcscence having excitaiion and emission maxima nt 328 and 406 nm. rcspectivcly. was formed specifically with uranic acids. This paper deals with a spectrofluorimetric method for the micro determination of uranic acids based on the latter reaction conditions. Under the neutral conditions employed, glycosidic linkages in aliphatic and aromatic uronides are not cleaved by hydrolysis. Therefore. unconjugated uranic acids can be determined nccurately without intcrfcrcnce from uronides.
Among
several
kinds
of buffer
solutions
I
I .Y
I
5
II
Fig. I. pH Dcpcndcncc of lluorcsccncc 2.OO*IO-J M: reaction time. 3 II. Fig. 2. Elkct reaction time.
having
of bulk 3 Ii.
concentration.
D-golacturonic
I’,
in 0.1
acid.
pH values.
I
,111
formalion
various
M
I
2
;ICC~;IIC
2.00~ IO-”
II.
,‘.I,
bufkr.
kf;
acetate
I
I 0..
I.
D-pulxturonic
LICCtiIIC bulkr.
11
wzid.
ptl
6.00:
S. HONDA,
200
K. SUDO.
K. KAKEI-11.
H. YUKI.
K. TAKIURA
buffer was found to give the most intense fluorescence at 328 (excitation)/405 nm (emission). In phosphate buffer another fluorescence was observed at 400/465 nm. as reported’. Figure 1 shows the pH dependence of fluorescence formation. The higher the pH of acetate buffer. the more intense the lluorescence:However. since very high pH values weaken the buffer effect, pH 6 was considered to be appropriate. Figure 2 shows the relationship between fluorescence intensity and the concentration of acetate buffer. The hyperbola indicates that fluorescence intensity decreased with increasing buffer concentrations. Therefore, a buffer concentration of 0.1 M was adopted in the standard procedure. Fluorescence formation was also dependent on the amount of the reagent if less than a IO-fold molar amount relative to uranic acids was present. but the use of larger excesses of the reagent gave a constant intensity of fluorescence. In the standard procedure the maximum concentration (2~ IO-’ M) of the reagent is used that allows a buffer effect, Heating almost
time The fluorescence intensity increased gradually for 3 h. Thereafter it. became constant. Accordingly. a heating time of 3 h was considered to be appropriate.
Fluorcscertce spectra und calibration The fluorescence spectrum for D-galacturonic acid obtained by the standard procedure is depicted in Fig. 3. There are observed excitation and emission maxima at 328 and 405 nm. respectively. D-Glucuronic acid as well as its &tone gave similar spectra. Calibration curves for D-galacturonic acid and D-glucuronic acid are shown in Fig. 4. The lines form gently sloping sigmoid curves which show linearity for sample concentrations of 5.0*10-“-1.5. IO-’ M and 2.0. lO-s-2.O. 10m4 M in both cases. The sensitivity of D-glucuronic acid was 82% lower than that of D-galacturonic acid. The low sensitivity is attributable to acid-&tone equilibration, since D-glucuronolactone gave the same intensity as D-glucuronic acid. D-Galacturonic acid does not equilibrate because it lacks the structural requirement' for
1.0 2. ” ._
‘2 0 ; ._
2 .” ._I
x
2
II.5
-
-11 SUU
Fig. 3. Fluoresccncc reaction time. 3 h.
4
spcclrum.
uu
D-pnlucturonic
1,111
ucid.
2.00. low4
M:
0.1 M ucctatc
buffer,
pH
G.00:
FLUORIMETRY
OF UNCONJUGATED
0,s
UROl’jIC
1.0
Fig. 4. Culibrution curves of uranic acids obtninod acid: (b) D-giucuronic ncid or D-lillcllronolactonc. TABLE
ACIDS
1.s
201
x
10.5
bl
by the stnndtird proccciurc.
(n) D-~iii~tcttlr~nic
I
REPRODUClBILlTY (5 dctcrmirwtions
OF DETERMINATION were done at cnch level.)
lactonization. Precision data for various concentrations of sample solutions are given in Table 1. The results indicate that this method is accurate and reproducible. EXPERIMENTAL
All reagents and samples of uranic acids were of reagent grade. Samples of uronides were kindly donated by Dr. K. Okui of Central Research Laboratories, Chugai Pll~~rm~~ceuticals Co.. Ltd. Fluorescence was measured with a Hitachi MPF-2A spectrofluorimetcr in l-cm quartz cells. To a buffered reagent solution (3.00 ml), prepared by mixing 2. lOs2 M ethylenediamine sulfate (I volume) and 0.1 M acetate buffer (pH 6.00, 2 volumes), add a sample solution (2.00 ml) containing 1.O* IO- z-3.0* IO- ’ or 4.0. IO- ‘-4.0 *IO- ’ llmofe of a uranic acid. Heat the mixture for 3 h on a boiling water bath. Determine the concentration of uranic acid by reading the fluorescence intensity at 328 (excitation) and 405 nm (emission) within 24 h. Prepare a calibration curve
S. HONDA,
202 simultaneously. distilled water. RESULTS
AND
A reagent
blank
is made
K. SUDO.
by
K. KAKEHI.
replacing
the
I-I. YUKI.
sample
K.TAKlURA
solution
with
DISCUSSION
Comparative studies under the standard reaction conditions indicated that these fluorescence reactions are highly selective for uranic acids. Various types of organic compounds cxamincd, including alcohols. phenols. umines. nitro compounds. kctoncs. carboxylic acids. esters. amides. nitrilcs, and sulfonic acids gave no fluorescence with ethylenediaminc sulfate. Some kinds of aromatic aldchydes, such as anisaldehyde and o-nitrobenzaldehyde. were the only exceptions. The relative fluorescence intensities at 328/405 nm. as referred to D-galacturonic acid, of all the 0.01 : aldoscs (glycol carbohydrates ( 1.O s IO- s M) listed below did not exceed aldehyde. DL-glyceraldehyde. D-erythrose. D-arab.inose. D-ribose. D-xylose. Dgalactose. D-glucose, D-mannose); ketoscs (D-fructose. L-sorbose); 2-deoxy sugars (2-deoxy-D-ribosc. 2-deoxy-D-glucose); methyl pcntoses (L-fucose. L-rhamnose); alditols (erythritol. D-xylitol. D-galactitol. D-mannitol. D-sorbitol): amino sugars (D-galactosamine hydrochloride. D-glucosamine hydrochloride. N-acetyl-D-galactosamine. N,-acetyl-D-glucosamine): aldonic acids (D-arabonic acid. D-gluconic acid); glycosides (methyl z- and /I-D-glucopyranosides. phenyl Q- and /3-D-glycopyranosides): oligosaccharides (maltose. cellobiose. lactose, sucrose, raffinose): polysacchitridcs (dcxtran. glycogcn. starch. chondroitin sulfates A and C). Determination of 1.0, lo-& M D-glucuronic acid was not affected by lo-fold molar amounts of D-glucose. but the prescncc of SL IOO-fold molar amount D-glucose caused considerable depression of fluorescence intensity for unknown reasons.
It is noticeable that. unlike acidic conditions, this fiuorimetric acids without cleaving glycosidic Accordingly, unconjugated uranic the presence of large amounts of TABLE
the calorimetric methodr-4 which USC strongly method makes it possible to determine uranic linkages in aliphatic and aromatic uronides. acids can bc determined without intcrfcrence in uronidcs. Uronides inspected include ethyl. n-
II
DETERMINATION
OF
D-GLUCURONIC
(In cuch cusc 2.00* lO-1 pmolc
D-.gIucumnic
ACID
IN THE
PRESENCE
OF D-GLUCURONIDES
acid wus used.)
Ethyl D-glucuronidc added (pmolc) * D-Glucuronic ocid found (pmolc~ 10bl)
0.20
0.40
0.60
0.80
I .M
I .2O
1.40
I.60
I.80
2.00
I .95
l.9H
2.0 I
I .9x
I .96
2.06
2.01
2.06
2.15
2.27
p-Tolyl D-glucuronidc uddcd (pmolc) D-Glucuronic acid round (pmolc. 10w2) --. -.___
0.20
0.40
0.60
0.x0
I.00
I .20
I .40
I .60
I.XO
2.00
I -94
I.96
I.98
I .97
I.98
‘*OS
2.07
2.06
2.18
2.25
_--.----
~--._.--_--.--
FLUORIMETRY
OF
UNCONJUGATED
URONIC
ACIDS
203
propyl. isobutyl. phenyl. p-tolyl. and p-chlorophcnyl glucuronides. Table II gives typical data obtnincd from ethyl and p-tolyl glucuronides. It is clear that 1.0. lo-” M D-glucuronic acid can be determined accurately in the presence of at least 80-fold molar amounts of D-glucuronides. The authors express their sincere gratitude to Dr. K. Okui for the gift of uronidc samples. The authors are also grateful to Chugai Pharmaceuticals Co.. Ltd. for financial support. SUMMARY
A simple method for the spectrofluorimetric determination of uranic acids is proposed. The fluorescence at 328 (cxcitution) and 405 nm (emission) formed by heating sumplc solutions in acetate buffer containing large amounts of cthylcncdiamine sulfntc. can be used to detcrminc 1.0. IO-“-3.0. 10m2 or 4.0. 10S2-4.0, lo- ’ /lmole of unconjugated uronic acids accurately. without intcrferencc from other carbohydrate materials. especially uronidcs. REFERENCES I S. Honcla. K. Kakimolo. K. Kakchi and K. Takiutx .‘lrlct/. Clrirtr. rlc./c/. 70 ( 1974) 133. 2 Z. Dischc. J. 13id. Chw.. I67 ( 1947) 180: .I. Rid. Clrotr.. 17 1 ( 1947) 725: J. Rio/. ( 1950) 4x9.
Chw..
183
