Use of Water-Soluble 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide for the Fluorescent Determination of Uranic Acids and Carboxylic Acids Mikihiko
and Eiji Ichishima Chemistry,
Reaction between glucuronic acid and l-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) was monitored by the o-phthalaldehyde (OPA) method, which was developed for the fluorescent assay of compounds containing an amino group. About 1 nmol of glucuronic acid was detected by this method. This EDC-OPA method was effective in detecting not only acidic sugar but also carboxylic acid. Although the sensitivity of the EDC-OPA method was somewhat lower than that of amino acid determination by OPA, a very simple and convenient assay was attained for compounds containing a carboxyl group. 0 1990 Academic Press, Inc.
Sheehan and Hess (1) and Khorana (2) found that dicyclohexylcarbodiimide (DCC)2 was a useful reagent in the formation of the peptide bond by activation of carboxyl group. The determination of free carboxylic acid was examined by the use of water-soluble carbodiimide (3,4). For example, Kasai et al. described the spectrometric determination of carboxylic acids with DCC by the formation of hydroxamic acids (5). The resulting ferric hydroxamate was measured at 525 nm, and a linear correlation was demonstrated for 0.25-2.5 pmol of benzoic acid. On the other hand, various fluorescent labeling reagents for the determination of carboxylic acids by high-performance liquid chromatography (HPLC) have been reported (6-8). Diazomethane (6) and bromomethyl derivatives (7) were known as highly reactive 1 Present address: National Food Research Institute, Tsukuba, Ibaraki 305, Japan. ’ Abbreviations used: DCC, dicyclohexylcarbodiimide; EDC, lethyl-3-(3-dimethylaminopropyl)carbodiimide; OPA, o-phthalaldehyde; TEMED, N,N,N’,iV’-tetramethylethylenediamine.
reagents for fluorescent labeling, whereas water-soluble carbodiimide was used to bind carboxylic acid to the fluorescent reagents (8). In most cases, labeling reagents were rather expensive and unstable for handling. Preparation of various fluorescent derivatives of neutral sugars and amino sugars have been reported for HPLC analysis (9-11). However, a limited number of methods were reported for the assay of uranic acid. During experiments for preparing fluorescent derivatives of uranic acid, we found that water-soluble lethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) reacted with uranic acids and produced some intermediate compounds. The intermediates containing an imino group were readily detected by the fluorescent reagent o-phthalaldehyde (OPA), which is known to be a highly sensitive reagent for the detection of amino acids (12). Therefore, a microscale determination of uronic acid was devised by the use of EDC and OPA reactions. In the present paper, we describe a simple and convenient method for the analysis of not only acidic sugars but also carboxylic acids.
Materials. Water-soluble carbodiimide EDC and OPA were products of Nacalai Tesque (Kyoto, Japan). Amberlite IR-120 was the product of Rohm & Haas Co. Uranic acids, acidic sugars, carboxylic acids, and other reagents were analytical grade. Reaction with EDC. A sample solution (25 ~1) containing uranic acid or carboxylic acid was mixed with a 4% EDC solution (25 ~1) freshly dissolved in 100 mM N,N,N’,iV’ - tetramethylethylenediamine - (TEMED) HCl buffer (pH 4.75) and incubated at 30°C for 30 min. A lo-p1 aliquot was subjected to reaction with OPA.
0003-269’7190 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
Reaction of water-soluble tentative structure for the fluorescent tion of compound III and OPA.
carbodiimide EDC. Inset is a product obtained by the reac-
Reaction with OPA. The OPA reagent was prepared according to Roth (12). Briefly, 1 ml of an OPA (10 mg/ ml in ethanol) solution and 1 ml of a 2-mercaptoethanol (5 pi/ml in ethanol) solution were mixed with 60 ml of 50 mM borate buffer (pH 9.5). This OPA reagent was prepared daily, while the latter two solutions were stable at -25°C over several weeks. To the lo-p1 sample obtained from the EDC reaction step was added 200 ~1 of the OPA reagent, and the resulting solution was mixed thoroughly with a Vortex mixer. Then, 400 ~1 of water was added, and the relative fluorescence was measured within 5-25 min. The fluorescence intensity was read by a fluorospectrometer (JASCO FP-550A, Nippon Bunko Co.) at excitation and emission wavelengths of X,, 340 nm and X,, 455 nm. The volume of the above sample (610 ~1) was sufficient for both 200-~1 and l.O-ml microcells with a suitable cell-folder. Column
Glucuronic acid (10 mg, 43 pmol) and EDC (40 mg, 209 pmol) were dissolved in 100 mM TEMED-HCl buffer, pH 4.75 (1 ml), and incubated at 30°C for 3 h. During the incubation, the pH of the reaction mixture was adjusted to 4.75 with 1 M HCl solution. The sample was diluted with 2 ml water and applied to a column of Amberlite IR-120. The column was eluted successively with water and 0.2 M NaOH. Unreacted glucuronic acid eluted at the breakthrough fraction was detected by the m-hydroxydiphenyl method (13). The OPA-reactive intermediates were detected by the OPA method described above. Glucuronic acid (O-50 pmol) Paper chromatography. and EDC (200 pmol) dissolved in 100 mM TEMED-HCl buffer (pH 4.75) were mixed and incubated at 30°C for 30 min. About lo-~1 portions of the reaction mixture were spotted on Toyo No. 50 filter paper (20 x 20 cm) and irrigated at 50°C for 3.5 h with 65% 1-propanol as the solvent. Spots of sugars were located by silver nitrate (14). Carboxyl groups were detected under uv light after spraying successively with 0.5% EDC in an ethanol and OPA solution.
Reaction of glucuronic acid with EDC. The reaction between a carboxyl group and EDC is known to produce three forms of intermediate, i.e., 0-acylisourea (III), Nacylurea (IV), and acid anhydride (VI), as shown in Scheme I. In the absence of suitable nucleophiles, these intermediates remained rather stable, which was confirmed by the paper chromatographic analysis of the products (data not shown). Although EDC itself has no proton accompanying the nitrogen atom, EDC is activated by protonation in aqueous medium. The course of the reaction of glucuronic acid with EDC could be monitored by the OPA method, which is known to be a very sensitive method for the fluorescent measurement of amino acids (12). As shown in Fig. 1, the fluorescence intensity of OPA increased with an increase in glucuronic acid concentration. Since the EDC concentration (final 2%) in the reaction mixture was constant, saturated curves for 13.3 and 20 pmol glucuranic acid might be ascribed to the deficiency of EDC concentration relative to the amount of carboxyl groups. Production of the intermediates proceeded rapidly and reached a stationary state within 30 min. The fluorescence intensity of glucuronic acid (250 ng to 2.5 pg) ranged between about 8 and 70, values which were reliable for the calibration curve (Fig. 2). Although the fluorescence intensities obtained with 50-250 ng of glucuronic acid were smaller than 8, a linear correlation was also observed for these low concentrations of glucuranic acid. As shown by the amide formation mechanism in Scheme I, production of an acid anhydride (VI) was accompanied by the formation of N,N’-dialkylurea. Since a 100~pmol solution of urea and dimethylurea gave very
FIG. 1. The course of the reaction of glucuronic acid and watersoluble carbodiimide EDC. Glucuronic acid, 0 (0). 6.7 (0), 13.3 (‘I), and 20 pmol (V), was mixed with an equal volume of 4% EDC and incubated at 30°C. For each 10 pl of the reaction mixture was subjected to the OPA method.
D-Glucuronic 50 loo ’ I
acid 150 I
(ng,o) 200 250 I 17
Responsesof Carboxylic Acids to the EDC-OPA Method Carboxylic Glyceric Glycolic Benzoic Malonic Malonic Succinic Palmitic
acid acid acid acid acid (Na acid acid
response 433 358 42 47 47 29 44
a For each, 5 nmol of carboxylic acid was used. * Value for glucuronic acid (shown in Table 1) was taken
FIG. 2. Response. of glucuronic acid to the EDC-OPA Glucuronic acid concentrations of 0.25-2.5 pg/tube (0) rig/tube (0) were measured.
method. and 50-250
poor fluorescence, N,N’-dialkylurea and N-acylurea (IV) seemed not to be reactive with OPA; therefore, Oacylisourea (III) was presumed to have an OPA-reactive imino group as shown in Scheme I, inset. Roth described the OPA method as sensitive enough to detect 1 nmol of alanine solution (12). The OPA method also has been used with the amino acid analyzer system (15,16), where 0.5 nmol of amino acids could be detected. Present results showed that at least 1 nmol of glucuronic acid was detected (Fig. 2). Response of uranic acids and carboxylic acids. Since the EDC-OPA method could be applicable to various acidic sugars containing a carboxyl group, fluorescent responses of seven carboxyl sugars were compared (Table 1). Each 5 nmol of sugar solution, except for arginic acid, was subjected to the analysis. The value of the relative fluorescence intensity of glucuronic acid was taken as 100%. Galacturonic acid and N-acetylneuraminic acid gave values 1.7-fold higher than that of
of Uranic Acids and Acidic Sugars to the EDC-OPA Method Relative
100 170 47 41 44 169 39
Glucuronic acid (Na salt) Galacturonic acid Gluconic acid (Ca salt) Glucono-b-lactone Mannono-1,4-lactone N-Acetylneuraminic acid Arainic acidh D For each, 5 nmol * 5 pg used.
glucuronic acid. In contrast, gluconic acid (Ca’+ salt), glucono-d-lactone, and mannono-I,4-lactone gave values of 41-47%. Moreover, arginic acid (5 pg) gave the smallest value of 39%. As shown in Table 2, the EDC-OPA method was applied to various carboxylic acids (5 nmol each). Among the seven acids tested, responses of glyceric acid and glycolic acid were 433 and 358%, respectively, which were 3- to 4-fold higher than that of glucuronic acid. Benzoic acid, malonic acid, and palmitic acid gave values of about 40%, whereas succinic acid gave a rather low value of 29%. These results show that the EDCOPA method is widely applicable to various compounds containing a carboxyl group. Differences in the fluorescence intensity among the acidic sugars and carboxylic acid would depend on the type of functional group neighboring the modified carboxy1 group. A relatively low response of dicarboxylic acids such as malonic acid and succinic acid might be explained by the change in the pK value after modification of the first carboxyl group; that is, the second carboxy1 group of dicarboxylic acids was hardly reactive with EDC. Chromatography of glucuronic acid-EDC adduct. By reaction with EDC, glucuronic acid loses its negative charge, and the resulting two intermediate forms (Oacylisourea (III) and N-acylurea (IV) shown in Scheme I) have a weak positive charge derived from the secondary amine. This was confirmed by column chromatography with Amberlite IR-120 (Fig. 3). At the breakthrough fraction, most of the unreacted glucuronic acid was eluted and was measured calorimetrically by the m-hydroxydiphenyl method (13). The column was then eluted with 0.2 M NaCl and the fluorescent peak was detected by the OPA method. This peak was not detected by the m-hydroxydiphenyl method, which suggested that the carboxyl group of glucuronic acid was substituted by EDC. Paper chromatographic analysis showed that three reaction products were formed by incubating 5-50 pmol glucuronic acid with 100 pmol EDC at 30°C for 30 min.
ponents should be removed by an appropriate method such as an ion-exchange column. However, compared with the conventional methods, the EDC-OPA method has several advantages, i.e., no requirement of a particular heating system and hazardous reagents. REFERENCES
Tube No.O.5 ml) FIG. 3. Elution pattern of the glucuronic acid-EDC complex from the Amberlite IR-120 column. The reaction mixture of 43 pmol glucuranic acid and 209 pmol EDC was applied to the column (1.5 X 5.0 cm) and eluted with water and 0.2 M NaOH solution. Each 1.5-ml fraction was collected.
Spots were detected by the alkaline silver nitrate dip procedure (14), which is based on reaction with reducing power. The failure to detect the unreacted glucuronic acid in the reaction mixture at the 50 pmol concentration showed that all of the uranic acid seemed to be derivatized by EDC. Moreover, various acidic sugars and carboxylic acids could be detected on paper chromatograms by spraying with EDC and OPA solutions. Light-blue fluorescent spots were visible under the uv light. Since the OPA method gave sufficient results in HPLC analysis of amino acids, the present EDC-OPA method would be also applicable to the HPLC postlabeling system for acidic sugars and carboxylic acids. This EDC-OPA method is subjected to interference from amino compounds in the sample. Thus, these com-
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