201,190-196
ANALYTICALBIOCHEMISTRY
(1992)
A Calorimetric Method for the Quantitation Acids and a Specific Assay for Galacturonic
of Uranic Acid
Kimberley A. Taylor and Jock G. Buchanan-Smith Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada Nl G 2 WI
Received November 19, 1991
A method of quantitating uranic acids and uranic acids from pectin in particular is described. The method uses carbazole in 80% sulfuric acid with borate ions added. The assay is carried out at 60°C. This assay has some cross reactivity with aldose sugars and must be timed precisely. A further method that is specific for galacturonic acid is also described. This method uses concentrated sulfuric acid and carbazole only. Of the biological substances tested, only formaldehyde and glyceraldehyde showed a reactivity of more than 10% that of galacturonic acid on a weight to weight basis. 0 1992
Academic
Press,
Inc.
Pectin is a heteropolysaccharide found in and between plant cell walls. The amount and composition of the polymer vary with the plant species but all pectins are based on a backbone of anhydrogalacturonic acid. Animal feedstuffs may contain pectins in quantities ranging from almost none to over 10% of the total dry matter (1). A simple quantitative method for estimating this fraction would be of benefit to the analysis of feed and forage. A calorimetric analysis of the galacturonic acid residues of the pectin fraction should provide the necessary information. Dische (2) developed a method of quantitating uranic acids using carbazole in sulfuric acid. The reaction showed little sensitivity to the common sugars, organic acids, or hydroxy acids. Several uranic acid polymers were shown to react according to their monomer composition. In a later paper, Dische (3) showed that by careful manipulation of temperature with less concentrated sulfuric acid, galacturonic acid could be distinguished from glucuronic acid and several polyuronides. This was due to the differential rate of formation of the furans involved in color development. The method involved a short incubation at 6O”C, addition of carbazole,
and incubation at room temperature with exact timing to the readings. Dekker and Richards (4) used this second assay system to investigate methods of digesting plant pectins to monomer levels. They used a correction formula to deal with the problem of increasing absorbance during the readings. These authors concluded that an enzymatic digestion of the pectin was necessary since prehydrolysis with acid resulted in lower than expected uranic acid concentrations. This might have been due to incomplete hydrolysis or to decarboxylation of the uranic acids. McComb and McCready (5) incubated the reagents in a boiling-water bath to completely convert galacturonic acid to 5-carboxy-2-formylfuran and then added carbazole and incubated at room temperature as was done by Dische (1). McComb and McCready investigated the effects of heating time, acid concentration, carbazole concentration, and specificity. They found that methylated galacturonic acid residues must be saponified to yield results similar to those for galacturonic acid. Bitter and Muir (6) added borate ions to the acid reagent and incubated reagents in a boiling-water bath to obtain immediate and maximum color intensity in the assay. This method required careful temperature control as the acid-borate reagent was mixed with the sample in water. Polyuronide samples were boiled at this point and again cooled to room temperature and the carbazole was added. The mix was boiled once more to develop the chromagen. In an effort to further simplify the assay we modified the Bitter and Muir assay reagents in order to avoid the need for temperature control. A further modification eliminated the need to boil the sample twice. Since this method still suffered from interference by neutral sugars and did not distinguish between galacturanic and glucuronic acids, the assay was further manipulated with respect to incubation temperature and acid concentration in order to improve specificity.
190 All
Copyright 0 1992 rights of reproduction
0003-2697/92 $3.00 by Academic Press, Inc. in any form reserved.
GALACTURONIC TABLE
Reactivity
ACID
RESULTS
1
of Other Substances in Acid-Borate at Different Temperatures
Reagent
Amount (/d
Glucuronic
acid
100
Glucose Fructose Xylose
200
Arabinose Rhamnose
100 100 100 100
100 200
Fucose
Mannose Myoinositol
100
60°C
165.0
18.7 18.6 10.4 5.3
21.0 12.0 12.0 7.0 3.5 3.0 9.0
0.6 5.5 nd”
Lactic acid Bovine serum albumin
100
nd
HCHO
500
10.0
50
95°C
136.4
1.5
0.4
1.0 3.0
1.3
n nd, not detected.
MATERIALS
AND
AND
DISCUSSION
All of the uranic acid methods described, including that of Bitter and Muir (6), involve adding the sugar in a fairly large volume of water to concentrated sulfuric acid. Unless this addition is carefully controlled, the rapid and extreme rise in temperature that results causes highly variable assay results. Another source of
Apparent galacturonate (%, w/w) Sample
191
ASSAY
METHODS
Chemicals used. Concentrated sulfuric acid (96%) was from Fisher Scientific. All other chemicals were from Sigma. The pectin used was from citrus (P-9135), and it is listed as containing approximately 85% galacturonic acid with a methoxy content of approximately 9.5%. Acid borate reagent. For 100 ml reagent, 0.80 g sodium borate (tetrahydrate) was dissolved in 16.67 ml water; 83.33 ml concentrated sulfuric acid was added carefully to form a layer. The bottle was allowed to remain undisturbed overnight to allow mixing without excessive heat production. Before this reagent was used it was checked to ensure that it was well mixed and had cooled to room temperature. This gives a sulfuric acid concentration of 80%. Carbazole reagent. Carbazole, 0.1% (w/v), was made up in absolute ethanol. &Zndard method. For a standard curve, 0 to 100 pg of galacturonic acid in up to 200 ~1 of water was added to a 16 X 150-mm borosilicate tube. Concentrated sulfuric acid (for galacturonic acid) or sulfuric acid-borate reagent (for uranic acids) (3 ml) was added. Carbazole reagent (100 ~1) was then added and the tube mixed. The tubes were then incubated at 60°C in a water bath for 1 h and cooled to room temperature in another water bath. The absorbance of the pink- to red-colored samples was read on a spectrophotometer at 530 nm against a water blank. If maximum discrimination between the uranic acids is required the incubation can be carried out at 40°C for 4 h.
variation
is the addition of the carbazole
reagent to the
sample before the acid is added. This results in precipitation
of the carbazole.
By premixing the water and acid and adding the sample in a small volume of water, the need to control temperature at this point was eliminated. Sugar in 100 ~1 water and 3 ml acid-borate reagent were incubated in a boiling-water bath (95°C) for 10 min and cooled to room temperature; 100 ~1 of 0.1% carbazole in ethanol was added and the tubes were boiled for a further 15 min. The method was found to be accurate and sensitive, with a linear curve of absorbance (530 nm) against sugar up to 100 pg. Twenty micrograms of galacturonic acid gives an absorbance of 0.400. Table 1 (the rightmost column) shows the reaction of several substances in the assay expressed as a percentage of apparent uranic acid on a weight by weight basis. The samples in 100 ~1 water were combined with 3 ml of acid-borate reagent and held in a boiling water bath (95°C) for 10 min. After being cooled to room temperature, 100 ~1 of 0.1% carbazole in absolute ethanol was added and the tubes were incubated once more. From these results it seemed likely that by reducing the temperature of incubation and using a single period of heating, a simpler assay that showed less interference from neutral sugars might be attained.
5 min 10 min ---*--. 20
min 0
30 n-l" -.-&-40 ml" -.__ *-.. 60 min -.-. * -.-.
0.2 1 0
I 20
Time
I 40
at
I 60
Room
I 80
Temperature
I 100
, 120
I 140
(min.)
FIG. 1. The change in absorbance over time at room temperature of the chromagen formed with galacturonic acid, 40 pg sugar, 3 ml acid-borate reagent, and 100 ~10.1% carbazole were mixed and incubated at 609C for 5, 10,20,40,60, and 90 min before being cooled to room temperature.
192
TAYLOR
AND
BUCHANAN-SMITH TABLE
Chromagen
Production
in Concentrated
2
Sulfuric
Acid Reagent
at Different
Temperature Amount (rg)
Sample
Room temperature
Temperatures (“C)
40
60
95
Galacturonic acid Glucuronic acid
100 100
0.684 0.049
(100”) (3.6)
1.567 0.076
(100) (2.4)
1.759 0.274
(100) (8)
0.809 0.249
(100) (15)
Glucose Fructose Sucrose Xylose
200 100 200 200
0.031 0.104 0.119 0.032
(2.3) (15) (8.7) (2.3)
0.072 0.118 0.145 0.034
(2.3) (7.5) (4.6) (1.1)
0.201 0.161 0.226 0.069
(5.7) (9.2) (6.4) (2)
0.370 0.197 0.360 0.069
(23) (24) (22) (4)
50 1000 74
0.027 0.027 0.606
(7.9) (0.4) (120)
0.034 0.028 0.689
(4.3) (0.2) (59)
0.042 0.030 0.888
(5) (0.2) (68)
0.034 0.036 1.126
(8) (0.4) (188)
Lactic Bovine HCHO
acid serum
D Absorbance
albumin
at 523 nm and the percentage
of galacturonic
acid absorbance
A 60°C incubation was used and pectin, galacturonic acid, and glucose were tested for absorbance over time. Fourty micrograms of sugar, 3 ml of acid-borate reagent, and 100 ~1 of 0.1% carbazole were incubated at 60°C for various times and read at A, nm. Both pectin and galacturonic acid completely reacted at 60 min. The absorbance for pectin was 112% that of the monomer. This compares to an expected value of 109% if the pectin had been completely hydrolysed to galacturonic acid residues. The maximum absorbance for 40 pg galacturanic acid achieved at 60°C is about 0.950 units; this compares to a value of 1.000 units for the boiling-water bath incubation. The absorbance due to glucose rose in
1.2
I
A la, F a, b 0.8 z.u ; 0.6 -
adjusted
to equal
weight
assuming
TABLE
-
3
galacturonate
(W, w/w)
Amount”
‘0 :: Q 0.2 0 300
response.
A Comparison of the Uranic Acid and Galacturonic Acid Methods Apparent
0.4
linear
a linear fashion during incubation at 60°C. At 5 min the glucose value was 5% and at 1 h it was 14% of the absorbance due to galacturonic acid on an equal weight basis. In many of the galacturonic acid assays, the absorbance continues to rise at room temperature throughout the time that the samples are being read. The boric acid in the Bitter and Muir assay (6) is said to eliminate this effect, the maximum absorbance being reached when the tubes are taken from the incubation bath. The effect of reducing the incubation temperature to 60°C on the chromagen development and stability was investigated. Figure 1 shows the effect of allowing the galacturonic acid chromagen to remain at room temperature after it was developed at 60°C for various times. After a 5-min incubation at 60°C the absorbance continues to rise at room temperature. After 2 h the absorbance is 166% of that immediately after cooling. This value is still less than one-half that obtained with a l-h incubation at
: :
value
Sample
(A%)
n-Arabinose I 400
I 500
I 600
Wavelength
700
800
900
1000
(nm)
FIG. 2. Absorption spectra of the chromagens produced using 60 pg of galacturonic acid. The first curve (solid line) was produced using 3 ml concentrated sulfuric acid and 100 pl incubation at 40°C temperature was used. was produced using the acid-borate reagent carbazole at 60°C temperature for 1.25 h of
of 0.1% carbazole. A 3-h For comparison a curve (3 ml) and 100 ~1 of 0.1% incubation (dashed line).
D-Fructose L-Fucose D-Glucose D-Ghrcuronic D-Mannose Myoinositol L-Rhamnose D-Xylose
acid
100 100 100 200 100 100 100 100 200
(1 Amount in parentheses * nd, not detected.
Uronate
(0.7) (0.6) (0.6) (1.1) (0.5) (0.6) (0.6) (0.6) (1.3) is in pmol.
(90 min) 5.3 18.6 0.6 18.7 100 5.5 nd 1.5 10.4
Galacturonate 4.0 9.2 nd* 5.7 8.0 5.3 nd nd 2.0
GALACTURONIC
ACID
193
ASSAY
1.4 6OC
6OC 60C 4OC
Galact. 1
Glut.
Galact. *
4ClC
Glut. *
40C
Galoct. . . . . a.-..
40C
acid
Galactwonic
SOC
acid
l-
borate
4OC 40C
z s
borate
0.8
-
0.6
-
0.4
-
0.2
-
Glucuronic
Golactwonic __L Gluctxonic . ..o
mid acid acid acid
a, ”
acid
C 0 D b LI)
Glut. ocld . . . . 0 _.._
2
0
100
50
Time
150
0*
200
0
(min.)
I 20
I 40
80
I 100
120
*cid6’(%)
FIG. 3. The time to maximum absorbance of 50 pg of sugar (in 100 pl water). Two reagents, concentrated acid and the acid-borate reagent, were used, as were two incubation temperatures, 4O’C (dashed lines) and 60°C (solid lines). Three milliliters of reagent and 100 al of 0.1% carbazole were added to all tubes.
FIG. 5. The effect of acid concentration on the absorbance of galacturonic acid and glucuronic acid when incubated at 40°C 4 h (dashed line) or at 60°C, 1 h (solid line). Below 64% acid the carbazole precipitated.
60°C. When the sugar is incubated for 1 h and then permitted to remain at room temperature, the absorbance drops at a rate of approximately 0.1% per minute. After a l-h incubation at 60°C the absorbance due to glucose did not change after remaining for a further 1 h at room temperature. The specificity of the reaction at 60°C was investigated. Table 1 shows the apparent uranic acid values for
several substances. The substances are tested in amounts much larger than would likely be encountered in typical sample mixtures. Values obtained using a 95°C incubation as described above are shown in the final column for comparison. Rhamnose and mannose were brown while fructose was orange in color. Formaldehyde was blue-green before heating. No other substance tested showed a color before heating. This modification of the Bitter and Muir assay was simpler to perform and took approximately the same amount of time but it offered little in the way of improved specificity for galacturonic over glucuronic acids. Table 2 shows the results of a screening test that was done to characterize the production of chromagen in concentrated sulfuric acid at various temperatures with a set of biological compounds. All incubations were for 1.5 h and the tubes were read immediately following. The assay mixtures consisted of test sample in 100 ~1 water, 3 ml concentrated acid, and 100 ~1 of 0.1% carbazole in absolute ethanol. The assays were read at an absorbance of 523 nm since that was found to be the maximum absorbing wavelength for the galacturonic acid. The formaldehyde sample had a maximum absorbance at 649 nm while the fructose sample absorbed maximally at 456 nm. The aldose sugars showed little color except at 95”C, where glucose showed more reactivity than xylose. As can readily be seen, galacturonic acid shows strong color development at 40 and 60°C while glucuronic acid shows little. It should also be noted that the galacturonic acid value is low at 95”C, perhaps indicating some destruction of the sugar. The absorbance spectrum (Fig. 2) for galacturonic acid using the 60°C incubation and the acid-borate re-
6OC 4Oc 4OC
,i 0
0
Gluturonic Calocturonic __.__... * Glucuronic .._ 0
acid acid acid
I I 0.5
I 1.5
I
Borax
(%
2
w/v)
FIG. 4. The effect of borate ion on the assay using 80% acid; 2.5 ml of acid and 0.5 ml of water containing varying amounts of borate were mixed and allowed to cool, and 100 pl of water containing galacturanic acid or glucuronic acid was added along with 100 pl of 0.1% carbazole. The tubes were incubated at 40°C for 3 h (dashed line) or 6O’C for 1 h (solid line). The usual acid-borate reagent contains 23.84 pg of sodium tetraborate decahydrate per assay tube. This amount is represented as the vertical dashed line in the figure.
194
TAYLOR TABLE
AND
4A
The Apparent Galacturonic Acid Content of Various stances and the Recovery of 20 pg Added Galacturonic Using the Concentrated Sulfuric Acid Reagent Apparent
SubAcid
galacturonate (%, w/w)
Amount (rmoU*
Galacturonic acid (1 h incubation)
Na acetate Na citrate Na citrate NaHCO, NaHPO, . 7H20 Tris . HCl
150 150 80 150 30 150
nd’ nd nd nd nd nd
103 51 32 100 97 105
Ethanol Methanol ~NHCI 1 N H.#O, NaEDTA
80 /.d 80 ~1 80 ~1 80 /d 1.37
nd nd nd nd nd
75 76 73 102 98
Sample”
(400)
Acetaldehyde Glyceraldehyde Benzaldehyde Formaldehyde Glutaraldehyde Hexanal (83) Octanal (82) Propionaldehyde
(39.4) (78.8) (50) (100) (105) (74) (100)
(81)
0.89 1.79 0.56 1.11 0.99 2.46 1.00 0.83 0.64 1.39
BUCHANAN-SMITH
3 7 39 155 nd 168 38 nd nd nd
Recovery (0.09 rmol)
or more) while glucuronic acid values rose some 2 to 8% under the same conditions. If maximum sensitivity is not a concern, there is potential to use a 95°C incubation for a very fast assay of galacturonic acid. The interference by true sugars and other substances in the sample would have to be investigated since their reactions in strong acids increase with increasing temperature. Table 2 gives some indication of the interference from other sugars that may be expected after 1.5 h in a boiling water bath using the concentrated acid reagent; shorter incubation times should improve on these values. The lower incubation temperatures were further in-
TABLE
Using
the
Concentrated
Sulfuric
73
Amount Sample0
a Amount in parentheses is in pg. ’ Unless otherwise specified. ’ nd, not detected.
agent was produced, along with a comparison spectrum for the concentrated acid method at 40°C. The peak for the concentrated acid test is shifted slightly to 523 nm but for the purposes of comparison, all chromagens were read at 530 nm. The acid-borate assay and the concentrated acid assay both produce equivalent absorbances for the same amount of sugar. The concentrated acid and acid-borate reagents were tested using a boiling-water bath incubation at 5,10,15, and 25 min. The acid reagent showed a maximum galacturanic acid absorbance after 5 min. This was unchanged with increasing time at 95°C and was also stable at room temperature for 4 h after the incubation. Glucuronic acid showed very low reactivity under the same conditions. Although this appeared to be a stable, specific, and fast assay, the absorbance achieved with galacturanic acid is less than 50% that achieved using a 40°C incubation temperature (see Table 2) or using the acidborate reagent. The acid-borate reagent showed a maximum absorbance for galacturonic and glucuronic acids at 10 min. Galacturonic acid absorbances showed a loss of about 2% over 4 h at room temperature (boiled 10 min
Acid
Amygdalin (50) D-Arabinose (50) D-Cellobiose (50) D-Fructose (50) L-Fucose (50) D-Glucose (50) Mannan (50) D-Mannose (50) Melibiose (50) D-Raffinose (50) L-Rhamnose (50) Rutin (50) D-XylOSe
(50)
D-Mannitol (50) Myoinositol (50) Sucrose (50) D-Galactonate (100) D-Gluconate (100) D-Glucuronate (50) Heparin (50) Pectin (50) Phytic acid (50) 6-Phosphogluconic acid (50) Polygalacturonate (50) D-Saccharate (100) D-Galactosamine (100) D-Glucosamine (100) D-Mannosamine (100)
(rcmoU*
SubAcid
Reagent
Apparent
91
-
4B
Apparent Galacturonic Acid Content of Various stances and the Recovery of 20 pg Added Galacturonic The
galacturonate (%, w/w)
Galacturonic acid (1 h incubation)
Recovery (0.09 pmol)
0.11 0.33 0.15 0.28 0.30 0.28 50 Pg 0.28 0.15 0.08 0.30 0.08 0.33 0.27 0.28 0.15
7.5 7 11 7 nd” 4 5.8 8 7 7 nd 2.8 4 nd nd
98 104 97 93 107 97 105 95 97 100 95 96 103 106 109 104
0.46 0.46 0.25 50 Prg 50 P&t 0.08
nd nd 5 19 51 nd
100 95 92 106 134 104
0.10
nd
103
50 Pg 0.40
80 1
163 105
0.46
1.5
100
0.46
1.5
95
0.46
2
’ Amount in parentheses is in pg. * Unless otherwise specified. ’ nd, not detected.
100
GALACTURONIC
vestigated. The acid-borate reagent, containing 80% sulfuric acid, and the concentrated sulfuric acid reagent were compared. Standard curves, relating the absorbance at 530 nm with respect to 0 to 100 pg (in 100 ~1 water) of either glucuronic or galacturonic acid, were made. The assays were carried out at either 60°C using 3 ml of acid-borate reagent (1.25 h incubation) or 40°C using concentrated acid reagent (3 h incubation). All assays used 100 ~1 of 0.1% carbazole in absolute ethanol. Galacturonic acid values were almost the same in both assays, the 40°C assay giving values about 10% lower than those of the 60°C assay. Glucuronic acid was detected with the acid-borate reagent and gave absorbance values about 25% greater than those for galacturanic acid. With the concentrated acid reagent the glucuronic acid absorbance remained almost absent. All curves were linear. The stability at room temperature of the 60-pug samples from these curves was investigated. The acid-borate assays showed some loss in absorbance while the concentrated acid reagent was stable over 21 h. The two sugars were assayed in the two reagents at two different temperatures in order to assay their times to maximum absorbance (Fig. 3). With the acid-borate reagent, galacturonic acid reaches a maximum absorbance at 40 min using a 60°C incubation. Glucuronic acid achieves this value at 60 min and is still rising after 180 min. In a 40°C incubation neither sugar reaches a maximum after 180 min. If the 60°C assay is stopped at 60 min the absorbance values remain stable. This allows the assumption that both sugars contribute equally to the absorbance. Using the concentrated acid reagent, glucuronic acid shows a small reaction at 60°C incubation, while at 40°C it is barely detectable. The galacturonic acid reaches a value at 60 min that is 90% of that reached at 180 min. At 40°C the same value is achieved in 180 min. At 60°C for 60 min the glucuronic acid absorbance is 6% that of the galacturonic acid. At 40°C and 3 h incubation the glucuronic acid absorbance is 4% that of galacturanic acid. When these assays were performed by removing the incubation tube, reading the absorbance, and then replacing the tube in the water bath to incubate further, it was noted that the 60°C incubation of galacturonic acid in the acid-borate reagent showed a distinct loss in absorbance over time after reaching the maximum. This was not seen if the tubes were read and then set aside rather than returned to the bath. This phenomenon is likely related to the solubility of the carbazole in 80% acid. The carbazole or the chromagen may precipitate when the solution cools while being measured and may not dissolve on reheating. Factors affecting the selectivity of the assay toward glucuronic and galacturonic acids were investigated. The effect of the borate ion in the 80% acid mixture is
ACID
ASSAY
195
shown in Fig. 4. Increasing the borate ion concentration causes a slight drop in the galacturonic acid absorbance while a large effect is noted on the glucuronic acid absorbance. Even with no borate ion present, glucuronic acid forms a chromagen at this acid concentration. Twenty-four micrograms of sodium tetraborate decahydrate per tube was added to concentrated acid reagent to test the effect of this ion. Samples were incubated at 40°C for 4 h or 60°C for 1 h with or without borate. Galacturonic and glucuronic acids as well as xylose and glucose were tested. Borate ions allowed the detection of glucuronic acid, but only to 50% of the absorbance obtained using the acid-borate reagent that contained 80% sulfuric acid. The absorbance was the same as that for galacturonic acid at 60°C but was only 48% of the absorbance in the 40°C incubation. Galacturanic acid values dropped 15 to 20% when borate was added. With added borate, glucose sugar showed an increase in absorbance from 4 to 19% of the absorbance obtained for galacturonic acid on an equal weight basis. Xylose was uniformly low in reaction under all reaction conditions. Figure 5 shows the effect of manipulating the acid concentration on the absorbance of galacturonic and glucuronic acid. Below 2 ml acid (64%) the carbazole precipitated. Maximum absorbance for galacturonic acid was at 2.75 ml acid (88%) but at this concentration glucuronic acid also reacts. When 3 ml acid (concentrated) was used the glucuronic acid values were 6 and 4% of those for galacturonate at 60 and 40°C respectively. The concentration of acid present in the acidborate reagent is represented at 2.5 ml or 80%. At this level the glucuronic acid absorbance is maximal but is about 20 and 30% that found using the acid-borate reagent at 40 and 60°C. In his original paper, Dische (2) used 82% sulfuric acid while in his modified method (3), he used 76%. Dekker and Richards (4) used 78% acid while McComb and McCready (5) recommended a concentration of 82%. All of these concentrations are prior to the addition of carbazole. In every case, glucuronic acid shows significant reaction in the assay. The effect of reduced carbazole concentration in the concentrated acid reagent assay was investigated. For galacturonic acid at either 40 or 60°C incubation the absorbance reached a plateau at 70 ~1 of 0.1% concentration. This was for 50 pg of sugar. Since the standard curves are linear to 100 pg sugar there would appear to be no problem with using 100 ~1 of carbazole in this assay. The concentrated acid assay at either 40 or 60°C shows no response to 200 pg of the following compounds: mannitol, rhamnose, myoinositol, arabinose, fucose, mannose, and tryptophan. Table 3 shows a comparison of the acid-borate and concentrated acid methods, as given in the methods section, for various sugars.
196
TAYLOR
AND BUCHANAN-SMITH
Tables 4a and 4b show absorbance values for various buffer salts, extractant reagents, and sugars tested with the concentrated acid, 60°C assay. Values for the recovery of added galacturonic acid are also given to identify possible interferances. Sodium citrate showed no absorbance alone but caused a lower recovery of galacturonic acid. EDTA did not show the same effect. Ethanol, methanol, and HCI all showed a drop in recovery of about 25%. Acetaldehyde had no effect while glyceraldehyde seemed to react somewhat with the carbazole. The sugars, sugar alcohols, and amino sugars all reacted at less than 10% in the assay and none interfered with the recovery of the added galacturonic acid. Among the acid sugars tested, only those containing glucuronic or galacturonic acid showed a reaction. Heparin, which contains glucuronic acid residues, reacted at 19% of the galacturonic acid value on a weight per weight basis. Pectin showed a 51% (w/w) value and polygalacturonic acid reacted at 80%. The pectin used here had a water content of 4.6%, a polygalacturonic acid content of 85%, and a methoxy content of 9.5%. Using these values, nonesterified galacturonic acid residues can be estimated at 65%. The polygalacturonic acid sugar is stated to be 85-90% de-esterified commercial pectin. Glucuronic acid, galacturonic acid, heparin, pectin, and polygalacturonic acid were all tested at 60°C in concentrated acid for 30 to 105 min in order to check whether the polymers were being completely broken
down. All sugars except pectin were essentially at their maximum value by 1 h. Pectin showed a maximum value of 66% at 1.5 h. Pectin, polygalacturonic acid, galacturonic acid, and glucuronic acid were assayed (6O”C, 1 h, concentrated acid) after saponification at room temperature for 30 min in 0.05 M NaOH. Values for unsaponified and saponified sugars respectively were galacturonic acid, 100 and 99%; glucuronic acid, 3 and 5%; polygalacturonic acid, 79 and 77%; and pectin, 52 and 79%. Only pectin was affected by saponification. The 60°C concentrated acid assay seems to be a suitable assay for selectively measuring galacturonic acid. Most of the other sugars tested with the assay show less than 10% reaction. Formaldehyde, glyceraldehyde, and sodium citrate will interfere with the detection of galacturanic acid. REFERENCES 1. Kennedy, J. F., and White, C. A. (1988) in Carbohydrate Chemistry (Kennedy, J. F., Ed.), pp. 220-261, Clarendon Press, Oxford. 2. Dische, Z. (1947) J. Biol. Chem. 16’7, 189-198. 3. Dische, Z. (1950) J. Viol. Chm. 183, 489-494. 4. Dekker, R. F. H., and Richards, G. N. (1972) J. Sci. Food Agric. 23,475-483.
McComb, E. A., and McCready, R. M. (1952) Anal. Chem. 1630-1632. 6. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4,330-334.
5.
24,
ANALYTICALBIOCHEMISTRY
(1992)
A Calorimetric Method for the Quantitation Acids and a Specific Assay for Galacturonic
of Uranic Acid
Kimberley A. Taylor and Jock G. Buchanan-Smith Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada Nl G 2 WI
Received November 19, 1991
A method of quantitating uranic acids and uranic acids from pectin in particular is described. The method uses carbazole in 80% sulfuric acid with borate ions added. The assay is carried out at 60°C. This assay has some cross reactivity with aldose sugars and must be timed precisely. A further method that is specific for galacturonic acid is also described. This method uses concentrated sulfuric acid and carbazole only. Of the biological substances tested, only formaldehyde and glyceraldehyde showed a reactivity of more than 10% that of galacturonic acid on a weight to weight basis. 0 1992
Academic
Press,
Inc.
Pectin is a heteropolysaccharide found in and between plant cell walls. The amount and composition of the polymer vary with the plant species but all pectins are based on a backbone of anhydrogalacturonic acid. Animal feedstuffs may contain pectins in quantities ranging from almost none to over 10% of the total dry matter (1). A simple quantitative method for estimating this fraction would be of benefit to the analysis of feed and forage. A calorimetric analysis of the galacturonic acid residues of the pectin fraction should provide the necessary information. Dische (2) developed a method of quantitating uranic acids using carbazole in sulfuric acid. The reaction showed little sensitivity to the common sugars, organic acids, or hydroxy acids. Several uranic acid polymers were shown to react according to their monomer composition. In a later paper, Dische (3) showed that by careful manipulation of temperature with less concentrated sulfuric acid, galacturonic acid could be distinguished from glucuronic acid and several polyuronides. This was due to the differential rate of formation of the furans involved in color development. The method involved a short incubation at 6O”C, addition of carbazole,
and incubation at room temperature with exact timing to the readings. Dekker and Richards (4) used this second assay system to investigate methods of digesting plant pectins to monomer levels. They used a correction formula to deal with the problem of increasing absorbance during the readings. These authors concluded that an enzymatic digestion of the pectin was necessary since prehydrolysis with acid resulted in lower than expected uranic acid concentrations. This might have been due to incomplete hydrolysis or to decarboxylation of the uranic acids. McComb and McCready (5) incubated the reagents in a boiling-water bath to completely convert galacturonic acid to 5-carboxy-2-formylfuran and then added carbazole and incubated at room temperature as was done by Dische (1). McComb and McCready investigated the effects of heating time, acid concentration, carbazole concentration, and specificity. They found that methylated galacturonic acid residues must be saponified to yield results similar to those for galacturonic acid. Bitter and Muir (6) added borate ions to the acid reagent and incubated reagents in a boiling-water bath to obtain immediate and maximum color intensity in the assay. This method required careful temperature control as the acid-borate reagent was mixed with the sample in water. Polyuronide samples were boiled at this point and again cooled to room temperature and the carbazole was added. The mix was boiled once more to develop the chromagen. In an effort to further simplify the assay we modified the Bitter and Muir assay reagents in order to avoid the need for temperature control. A further modification eliminated the need to boil the sample twice. Since this method still suffered from interference by neutral sugars and did not distinguish between galacturanic and glucuronic acids, the assay was further manipulated with respect to incubation temperature and acid concentration in order to improve specificity.
190 All
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GALACTURONIC TABLE
Reactivity
ACID
RESULTS
1
of Other Substances in Acid-Borate at Different Temperatures
Reagent
Amount (/d
Glucuronic
acid
100
Glucose Fructose Xylose
200
Arabinose Rhamnose
100 100 100 100
100 200
Fucose
Mannose Myoinositol
100
60°C
165.0
18.7 18.6 10.4 5.3
21.0 12.0 12.0 7.0 3.5 3.0 9.0
0.6 5.5 nd”
Lactic acid Bovine serum albumin
100
nd
HCHO
500
10.0
50
95°C
136.4
1.5
0.4
1.0 3.0
1.3
n nd, not detected.
MATERIALS
AND
AND
DISCUSSION
All of the uranic acid methods described, including that of Bitter and Muir (6), involve adding the sugar in a fairly large volume of water to concentrated sulfuric acid. Unless this addition is carefully controlled, the rapid and extreme rise in temperature that results causes highly variable assay results. Another source of
Apparent galacturonate (%, w/w) Sample
191
ASSAY
METHODS
Chemicals used. Concentrated sulfuric acid (96%) was from Fisher Scientific. All other chemicals were from Sigma. The pectin used was from citrus (P-9135), and it is listed as containing approximately 85% galacturonic acid with a methoxy content of approximately 9.5%. Acid borate reagent. For 100 ml reagent, 0.80 g sodium borate (tetrahydrate) was dissolved in 16.67 ml water; 83.33 ml concentrated sulfuric acid was added carefully to form a layer. The bottle was allowed to remain undisturbed overnight to allow mixing without excessive heat production. Before this reagent was used it was checked to ensure that it was well mixed and had cooled to room temperature. This gives a sulfuric acid concentration of 80%. Carbazole reagent. Carbazole, 0.1% (w/v), was made up in absolute ethanol. &Zndard method. For a standard curve, 0 to 100 pg of galacturonic acid in up to 200 ~1 of water was added to a 16 X 150-mm borosilicate tube. Concentrated sulfuric acid (for galacturonic acid) or sulfuric acid-borate reagent (for uranic acids) (3 ml) was added. Carbazole reagent (100 ~1) was then added and the tube mixed. The tubes were then incubated at 60°C in a water bath for 1 h and cooled to room temperature in another water bath. The absorbance of the pink- to red-colored samples was read on a spectrophotometer at 530 nm against a water blank. If maximum discrimination between the uranic acids is required the incubation can be carried out at 40°C for 4 h.
variation
is the addition of the carbazole
reagent to the
sample before the acid is added. This results in precipitation
of the carbazole.
By premixing the water and acid and adding the sample in a small volume of water, the need to control temperature at this point was eliminated. Sugar in 100 ~1 water and 3 ml acid-borate reagent were incubated in a boiling-water bath (95°C) for 10 min and cooled to room temperature; 100 ~1 of 0.1% carbazole in ethanol was added and the tubes were boiled for a further 15 min. The method was found to be accurate and sensitive, with a linear curve of absorbance (530 nm) against sugar up to 100 pg. Twenty micrograms of galacturonic acid gives an absorbance of 0.400. Table 1 (the rightmost column) shows the reaction of several substances in the assay expressed as a percentage of apparent uranic acid on a weight by weight basis. The samples in 100 ~1 water were combined with 3 ml of acid-borate reagent and held in a boiling water bath (95°C) for 10 min. After being cooled to room temperature, 100 ~1 of 0.1% carbazole in absolute ethanol was added and the tubes were incubated once more. From these results it seemed likely that by reducing the temperature of incubation and using a single period of heating, a simpler assay that showed less interference from neutral sugars might be attained.
5 min 10 min ---*--. 20
min 0
30 n-l" -.-&-40 ml" -.__ *-.. 60 min -.-. * -.-.
0.2 1 0
I 20
Time
I 40
at
I 60
Room
I 80
Temperature
I 100
, 120
I 140
(min.)
FIG. 1. The change in absorbance over time at room temperature of the chromagen formed with galacturonic acid, 40 pg sugar, 3 ml acid-borate reagent, and 100 ~10.1% carbazole were mixed and incubated at 609C for 5, 10,20,40,60, and 90 min before being cooled to room temperature.
192
TAYLOR
AND
BUCHANAN-SMITH TABLE
Chromagen
Production
in Concentrated
2
Sulfuric
Acid Reagent
at Different
Temperature Amount (rg)
Sample
Room temperature
Temperatures (“C)
40
60
95
Galacturonic acid Glucuronic acid
100 100
0.684 0.049
(100”) (3.6)
1.567 0.076
(100) (2.4)
1.759 0.274
(100) (8)
0.809 0.249
(100) (15)
Glucose Fructose Sucrose Xylose
200 100 200 200
0.031 0.104 0.119 0.032
(2.3) (15) (8.7) (2.3)
0.072 0.118 0.145 0.034
(2.3) (7.5) (4.6) (1.1)
0.201 0.161 0.226 0.069
(5.7) (9.2) (6.4) (2)
0.370 0.197 0.360 0.069
(23) (24) (22) (4)
50 1000 74
0.027 0.027 0.606
(7.9) (0.4) (120)
0.034 0.028 0.689
(4.3) (0.2) (59)
0.042 0.030 0.888
(5) (0.2) (68)
0.034 0.036 1.126
(8) (0.4) (188)
Lactic Bovine HCHO
acid serum
D Absorbance
albumin
at 523 nm and the percentage
of galacturonic
acid absorbance
A 60°C incubation was used and pectin, galacturonic acid, and glucose were tested for absorbance over time. Fourty micrograms of sugar, 3 ml of acid-borate reagent, and 100 ~1 of 0.1% carbazole were incubated at 60°C for various times and read at A, nm. Both pectin and galacturonic acid completely reacted at 60 min. The absorbance for pectin was 112% that of the monomer. This compares to an expected value of 109% if the pectin had been completely hydrolysed to galacturonic acid residues. The maximum absorbance for 40 pg galacturanic acid achieved at 60°C is about 0.950 units; this compares to a value of 1.000 units for the boiling-water bath incubation. The absorbance due to glucose rose in
1.2
I
A la, F a, b 0.8 z.u ; 0.6 -
adjusted
to equal
weight
assuming
TABLE
-
3
galacturonate
(W, w/w)
Amount”
‘0 :: Q 0.2 0 300
response.
A Comparison of the Uranic Acid and Galacturonic Acid Methods Apparent
0.4
linear
a linear fashion during incubation at 60°C. At 5 min the glucose value was 5% and at 1 h it was 14% of the absorbance due to galacturonic acid on an equal weight basis. In many of the galacturonic acid assays, the absorbance continues to rise at room temperature throughout the time that the samples are being read. The boric acid in the Bitter and Muir assay (6) is said to eliminate this effect, the maximum absorbance being reached when the tubes are taken from the incubation bath. The effect of reducing the incubation temperature to 60°C on the chromagen development and stability was investigated. Figure 1 shows the effect of allowing the galacturonic acid chromagen to remain at room temperature after it was developed at 60°C for various times. After a 5-min incubation at 60°C the absorbance continues to rise at room temperature. After 2 h the absorbance is 166% of that immediately after cooling. This value is still less than one-half that obtained with a l-h incubation at
: :
value
Sample
(A%)
n-Arabinose I 400
I 500
I 600
Wavelength
700
800
900
1000
(nm)
FIG. 2. Absorption spectra of the chromagens produced using 60 pg of galacturonic acid. The first curve (solid line) was produced using 3 ml concentrated sulfuric acid and 100 pl incubation at 40°C temperature was used. was produced using the acid-borate reagent carbazole at 60°C temperature for 1.25 h of
of 0.1% carbazole. A 3-h For comparison a curve (3 ml) and 100 ~1 of 0.1% incubation (dashed line).
D-Fructose L-Fucose D-Glucose D-Ghrcuronic D-Mannose Myoinositol L-Rhamnose D-Xylose
acid
100 100 100 200 100 100 100 100 200
(1 Amount in parentheses * nd, not detected.
Uronate
(0.7) (0.6) (0.6) (1.1) (0.5) (0.6) (0.6) (0.6) (1.3) is in pmol.
(90 min) 5.3 18.6 0.6 18.7 100 5.5 nd 1.5 10.4
Galacturonate 4.0 9.2 nd* 5.7 8.0 5.3 nd nd 2.0
GALACTURONIC
ACID
193
ASSAY
1.4 6OC
6OC 60C 4OC
Galact. 1
Glut.
Galact. *
4ClC
Glut. *
40C
Galoct. . . . . a.-..
40C
acid
Galactwonic
SOC
acid
l-
borate
4OC 40C
z s
borate
0.8
-
0.6
-
0.4
-
0.2
-
Glucuronic
Golactwonic __L Gluctxonic . ..o
mid acid acid acid
a, ”
acid
C 0 D b LI)
Glut. ocld . . . . 0 _.._
2
0
100
50
Time
150
0*
200
0
(min.)
I 20
I 40
80
I 100
120
*cid6’(%)
FIG. 3. The time to maximum absorbance of 50 pg of sugar (in 100 pl water). Two reagents, concentrated acid and the acid-borate reagent, were used, as were two incubation temperatures, 4O’C (dashed lines) and 60°C (solid lines). Three milliliters of reagent and 100 al of 0.1% carbazole were added to all tubes.
FIG. 5. The effect of acid concentration on the absorbance of galacturonic acid and glucuronic acid when incubated at 40°C 4 h (dashed line) or at 60°C, 1 h (solid line). Below 64% acid the carbazole precipitated.
60°C. When the sugar is incubated for 1 h and then permitted to remain at room temperature, the absorbance drops at a rate of approximately 0.1% per minute. After a l-h incubation at 60°C the absorbance due to glucose did not change after remaining for a further 1 h at room temperature. The specificity of the reaction at 60°C was investigated. Table 1 shows the apparent uranic acid values for
several substances. The substances are tested in amounts much larger than would likely be encountered in typical sample mixtures. Values obtained using a 95°C incubation as described above are shown in the final column for comparison. Rhamnose and mannose were brown while fructose was orange in color. Formaldehyde was blue-green before heating. No other substance tested showed a color before heating. This modification of the Bitter and Muir assay was simpler to perform and took approximately the same amount of time but it offered little in the way of improved specificity for galacturonic over glucuronic acids. Table 2 shows the results of a screening test that was done to characterize the production of chromagen in concentrated sulfuric acid at various temperatures with a set of biological compounds. All incubations were for 1.5 h and the tubes were read immediately following. The assay mixtures consisted of test sample in 100 ~1 water, 3 ml concentrated acid, and 100 ~1 of 0.1% carbazole in absolute ethanol. The assays were read at an absorbance of 523 nm since that was found to be the maximum absorbing wavelength for the galacturonic acid. The formaldehyde sample had a maximum absorbance at 649 nm while the fructose sample absorbed maximally at 456 nm. The aldose sugars showed little color except at 95”C, where glucose showed more reactivity than xylose. As can readily be seen, galacturonic acid shows strong color development at 40 and 60°C while glucuronic acid shows little. It should also be noted that the galacturonic acid value is low at 95”C, perhaps indicating some destruction of the sugar. The absorbance spectrum (Fig. 2) for galacturonic acid using the 60°C incubation and the acid-borate re-
6OC 4Oc 4OC
,i 0
0
Gluturonic Calocturonic __.__... * Glucuronic .._ 0
acid acid acid
I I 0.5
I 1.5
I
Borax
(%
2
w/v)
FIG. 4. The effect of borate ion on the assay using 80% acid; 2.5 ml of acid and 0.5 ml of water containing varying amounts of borate were mixed and allowed to cool, and 100 pl of water containing galacturanic acid or glucuronic acid was added along with 100 pl of 0.1% carbazole. The tubes were incubated at 40°C for 3 h (dashed line) or 6O’C for 1 h (solid line). The usual acid-borate reagent contains 23.84 pg of sodium tetraborate decahydrate per assay tube. This amount is represented as the vertical dashed line in the figure.
194
TAYLOR TABLE
AND
4A
The Apparent Galacturonic Acid Content of Various stances and the Recovery of 20 pg Added Galacturonic Using the Concentrated Sulfuric Acid Reagent Apparent
SubAcid
galacturonate (%, w/w)
Amount (rmoU*
Galacturonic acid (1 h incubation)
Na acetate Na citrate Na citrate NaHCO, NaHPO, . 7H20 Tris . HCl
150 150 80 150 30 150
nd’ nd nd nd nd nd
103 51 32 100 97 105
Ethanol Methanol ~NHCI 1 N H.#O, NaEDTA
80 /.d 80 ~1 80 ~1 80 /d 1.37
nd nd nd nd nd
75 76 73 102 98
Sample”
(400)
Acetaldehyde Glyceraldehyde Benzaldehyde Formaldehyde Glutaraldehyde Hexanal (83) Octanal (82) Propionaldehyde
(39.4) (78.8) (50) (100) (105) (74) (100)
(81)
0.89 1.79 0.56 1.11 0.99 2.46 1.00 0.83 0.64 1.39
BUCHANAN-SMITH
3 7 39 155 nd 168 38 nd nd nd
Recovery (0.09 rmol)
or more) while glucuronic acid values rose some 2 to 8% under the same conditions. If maximum sensitivity is not a concern, there is potential to use a 95°C incubation for a very fast assay of galacturonic acid. The interference by true sugars and other substances in the sample would have to be investigated since their reactions in strong acids increase with increasing temperature. Table 2 gives some indication of the interference from other sugars that may be expected after 1.5 h in a boiling water bath using the concentrated acid reagent; shorter incubation times should improve on these values. The lower incubation temperatures were further in-
TABLE
Using
the
Concentrated
Sulfuric
73
Amount Sample0
a Amount in parentheses is in pg. ’ Unless otherwise specified. ’ nd, not detected.
agent was produced, along with a comparison spectrum for the concentrated acid method at 40°C. The peak for the concentrated acid test is shifted slightly to 523 nm but for the purposes of comparison, all chromagens were read at 530 nm. The acid-borate assay and the concentrated acid assay both produce equivalent absorbances for the same amount of sugar. The concentrated acid and acid-borate reagents were tested using a boiling-water bath incubation at 5,10,15, and 25 min. The acid reagent showed a maximum galacturanic acid absorbance after 5 min. This was unchanged with increasing time at 95°C and was also stable at room temperature for 4 h after the incubation. Glucuronic acid showed very low reactivity under the same conditions. Although this appeared to be a stable, specific, and fast assay, the absorbance achieved with galacturanic acid is less than 50% that achieved using a 40°C incubation temperature (see Table 2) or using the acidborate reagent. The acid-borate reagent showed a maximum absorbance for galacturonic and glucuronic acids at 10 min. Galacturonic acid absorbances showed a loss of about 2% over 4 h at room temperature (boiled 10 min
Acid
Amygdalin (50) D-Arabinose (50) D-Cellobiose (50) D-Fructose (50) L-Fucose (50) D-Glucose (50) Mannan (50) D-Mannose (50) Melibiose (50) D-Raffinose (50) L-Rhamnose (50) Rutin (50) D-XylOSe
(50)
D-Mannitol (50) Myoinositol (50) Sucrose (50) D-Galactonate (100) D-Gluconate (100) D-Glucuronate (50) Heparin (50) Pectin (50) Phytic acid (50) 6-Phosphogluconic acid (50) Polygalacturonate (50) D-Saccharate (100) D-Galactosamine (100) D-Glucosamine (100) D-Mannosamine (100)
(rcmoU*
SubAcid
Reagent
Apparent
91
-
4B
Apparent Galacturonic Acid Content of Various stances and the Recovery of 20 pg Added Galacturonic The
galacturonate (%, w/w)
Galacturonic acid (1 h incubation)
Recovery (0.09 pmol)
0.11 0.33 0.15 0.28 0.30 0.28 50 Pg 0.28 0.15 0.08 0.30 0.08 0.33 0.27 0.28 0.15
7.5 7 11 7 nd” 4 5.8 8 7 7 nd 2.8 4 nd nd
98 104 97 93 107 97 105 95 97 100 95 96 103 106 109 104
0.46 0.46 0.25 50 Prg 50 P&t 0.08
nd nd 5 19 51 nd
100 95 92 106 134 104
0.10
nd
103
50 Pg 0.40
80 1
163 105
0.46
1.5
100
0.46
1.5
95
0.46
2
’ Amount in parentheses is in pg. * Unless otherwise specified. ’ nd, not detected.
100
GALACTURONIC
vestigated. The acid-borate reagent, containing 80% sulfuric acid, and the concentrated sulfuric acid reagent were compared. Standard curves, relating the absorbance at 530 nm with respect to 0 to 100 pg (in 100 ~1 water) of either glucuronic or galacturonic acid, were made. The assays were carried out at either 60°C using 3 ml of acid-borate reagent (1.25 h incubation) or 40°C using concentrated acid reagent (3 h incubation). All assays used 100 ~1 of 0.1% carbazole in absolute ethanol. Galacturonic acid values were almost the same in both assays, the 40°C assay giving values about 10% lower than those of the 60°C assay. Glucuronic acid was detected with the acid-borate reagent and gave absorbance values about 25% greater than those for galacturanic acid. With the concentrated acid reagent the glucuronic acid absorbance remained almost absent. All curves were linear. The stability at room temperature of the 60-pug samples from these curves was investigated. The acid-borate assays showed some loss in absorbance while the concentrated acid reagent was stable over 21 h. The two sugars were assayed in the two reagents at two different temperatures in order to assay their times to maximum absorbance (Fig. 3). With the acid-borate reagent, galacturonic acid reaches a maximum absorbance at 40 min using a 60°C incubation. Glucuronic acid achieves this value at 60 min and is still rising after 180 min. In a 40°C incubation neither sugar reaches a maximum after 180 min. If the 60°C assay is stopped at 60 min the absorbance values remain stable. This allows the assumption that both sugars contribute equally to the absorbance. Using the concentrated acid reagent, glucuronic acid shows a small reaction at 60°C incubation, while at 40°C it is barely detectable. The galacturonic acid reaches a value at 60 min that is 90% of that reached at 180 min. At 40°C the same value is achieved in 180 min. At 60°C for 60 min the glucuronic acid absorbance is 6% that of the galacturonic acid. At 40°C and 3 h incubation the glucuronic acid absorbance is 4% that of galacturanic acid. When these assays were performed by removing the incubation tube, reading the absorbance, and then replacing the tube in the water bath to incubate further, it was noted that the 60°C incubation of galacturonic acid in the acid-borate reagent showed a distinct loss in absorbance over time after reaching the maximum. This was not seen if the tubes were read and then set aside rather than returned to the bath. This phenomenon is likely related to the solubility of the carbazole in 80% acid. The carbazole or the chromagen may precipitate when the solution cools while being measured and may not dissolve on reheating. Factors affecting the selectivity of the assay toward glucuronic and galacturonic acids were investigated. The effect of the borate ion in the 80% acid mixture is
ACID
ASSAY
195
shown in Fig. 4. Increasing the borate ion concentration causes a slight drop in the galacturonic acid absorbance while a large effect is noted on the glucuronic acid absorbance. Even with no borate ion present, glucuronic acid forms a chromagen at this acid concentration. Twenty-four micrograms of sodium tetraborate decahydrate per tube was added to concentrated acid reagent to test the effect of this ion. Samples were incubated at 40°C for 4 h or 60°C for 1 h with or without borate. Galacturonic and glucuronic acids as well as xylose and glucose were tested. Borate ions allowed the detection of glucuronic acid, but only to 50% of the absorbance obtained using the acid-borate reagent that contained 80% sulfuric acid. The absorbance was the same as that for galacturonic acid at 60°C but was only 48% of the absorbance in the 40°C incubation. Galacturanic acid values dropped 15 to 20% when borate was added. With added borate, glucose sugar showed an increase in absorbance from 4 to 19% of the absorbance obtained for galacturonic acid on an equal weight basis. Xylose was uniformly low in reaction under all reaction conditions. Figure 5 shows the effect of manipulating the acid concentration on the absorbance of galacturonic and glucuronic acid. Below 2 ml acid (64%) the carbazole precipitated. Maximum absorbance for galacturonic acid was at 2.75 ml acid (88%) but at this concentration glucuronic acid also reacts. When 3 ml acid (concentrated) was used the glucuronic acid values were 6 and 4% of those for galacturonate at 60 and 40°C respectively. The concentration of acid present in the acidborate reagent is represented at 2.5 ml or 80%. At this level the glucuronic acid absorbance is maximal but is about 20 and 30% that found using the acid-borate reagent at 40 and 60°C. In his original paper, Dische (2) used 82% sulfuric acid while in his modified method (3), he used 76%. Dekker and Richards (4) used 78% acid while McComb and McCready (5) recommended a concentration of 82%. All of these concentrations are prior to the addition of carbazole. In every case, glucuronic acid shows significant reaction in the assay. The effect of reduced carbazole concentration in the concentrated acid reagent assay was investigated. For galacturonic acid at either 40 or 60°C incubation the absorbance reached a plateau at 70 ~1 of 0.1% concentration. This was for 50 pg of sugar. Since the standard curves are linear to 100 pg sugar there would appear to be no problem with using 100 ~1 of carbazole in this assay. The concentrated acid assay at either 40 or 60°C shows no response to 200 pg of the following compounds: mannitol, rhamnose, myoinositol, arabinose, fucose, mannose, and tryptophan. Table 3 shows a comparison of the acid-borate and concentrated acid methods, as given in the methods section, for various sugars.
196
TAYLOR
AND BUCHANAN-SMITH
Tables 4a and 4b show absorbance values for various buffer salts, extractant reagents, and sugars tested with the concentrated acid, 60°C assay. Values for the recovery of added galacturonic acid are also given to identify possible interferances. Sodium citrate showed no absorbance alone but caused a lower recovery of galacturonic acid. EDTA did not show the same effect. Ethanol, methanol, and HCI all showed a drop in recovery of about 25%. Acetaldehyde had no effect while glyceraldehyde seemed to react somewhat with the carbazole. The sugars, sugar alcohols, and amino sugars all reacted at less than 10% in the assay and none interfered with the recovery of the added galacturonic acid. Among the acid sugars tested, only those containing glucuronic or galacturonic acid showed a reaction. Heparin, which contains glucuronic acid residues, reacted at 19% of the galacturonic acid value on a weight per weight basis. Pectin showed a 51% (w/w) value and polygalacturonic acid reacted at 80%. The pectin used here had a water content of 4.6%, a polygalacturonic acid content of 85%, and a methoxy content of 9.5%. Using these values, nonesterified galacturonic acid residues can be estimated at 65%. The polygalacturonic acid sugar is stated to be 85-90% de-esterified commercial pectin. Glucuronic acid, galacturonic acid, heparin, pectin, and polygalacturonic acid were all tested at 60°C in concentrated acid for 30 to 105 min in order to check whether the polymers were being completely broken
down. All sugars except pectin were essentially at their maximum value by 1 h. Pectin showed a maximum value of 66% at 1.5 h. Pectin, polygalacturonic acid, galacturonic acid, and glucuronic acid were assayed (6O”C, 1 h, concentrated acid) after saponification at room temperature for 30 min in 0.05 M NaOH. Values for unsaponified and saponified sugars respectively were galacturonic acid, 100 and 99%; glucuronic acid, 3 and 5%; polygalacturonic acid, 79 and 77%; and pectin, 52 and 79%. Only pectin was affected by saponification. The 60°C concentrated acid assay seems to be a suitable assay for selectively measuring galacturonic acid. Most of the other sugars tested with the assay show less than 10% reaction. Formaldehyde, glyceraldehyde, and sodium citrate will interfere with the detection of galacturanic acid. REFERENCES 1. Kennedy, J. F., and White, C. A. (1988) in Carbohydrate Chemistry (Kennedy, J. F., Ed.), pp. 220-261, Clarendon Press, Oxford. 2. Dische, Z. (1947) J. Biol. Chem. 16’7, 189-198. 3. Dische, Z. (1950) J. Viol. Chm. 183, 489-494. 4. Dekker, R. F. H., and Richards, G. N. (1972) J. Sci. Food Agric. 23,475-483.
McComb, E. A., and McCready, R. M. (1952) Anal. Chem. 1630-1632. 6. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4,330-334.
5.
24,
