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Methods for determination of carbonyl compounds by 2,4-dinitrophenylhydrazine and their application to the assay of aldehyde dehydrogenase
ANALYTICAL
Methods
BIOCHEMISTRY
for
43, 446-453
(1971)
Determination
of
2,4-Dinitrophenylhydrazine to the Assay
of Chemistry,
Compounds
and
Application
of Aldehyde NAKAO
Department
Carbonyl Their
by
Dehydrogenase
ARIGA
Faculty of General Gif u, Japan
Education,
Gifu
University,
Received January 20, 1971
The determination of small amounts of carbonyl compounds in biological materials is of great importance for biochemical studies. Although many methods have been reported, the 2,4-dinitrophenylhydrazine method has been used most widely. For the determination of keto acids, the method of Friedemann and Haugen is well known (1). According to their method, the 2,4-dinitrophenylhydrazone formed by reaction with keto acid is extracted with an organic solvent from a water solution, retransferred into carbonate solution for separation from the unreacted reagent, and determined spectrophotometrically after addition of alkali. For a special sample containing a single keto acid as carbonyl compound, they proposed a direct method in which the determination was carried out omitting the extraction procedure. However, it was pointed out by Katsuki et al. that spectrophotometric measurement must be carried out under specific conditions because of the distinct molecular absorption spectra of the two geometric isomers of the hydrazone (2). From a consideration of this point, Katsuki et al. devised an improved direct method for determination of keto acids, and found that it gives satisfactory results (3). Unlike keto acids, the hydrazone of aldehyde or ketone, in general, gives one isomer under the determination conditions and consequently special consideration as to the wavelength for the determination is unnecessary. However, during studies on the determination of carbonyl compounds, the author with his collaborators pointed out the necessity for use of a special coefficient of hydrazone formation for the determination of some compounds because they form the hydrazones nonquantitatively but to a limited extent under the conditions commonly used (4). Furthermore, it was pointed out that a correction due to the coloration of the 446
DETERMINATION
OF
CARBONYL
COMPOUNDS
447
hydrazones of the carbonyl compounds present as impurities in alcohol used as solvent is necessary for the determination (4). The present paper deals with two methods devised for the determination of small amounts of aldehydes and ketones and their application to the assay of aldehyde dehydrogenase. REAGENTS
AND
MATERIALS
2,-J-Dinitrophenylhydrazine reagent. 2.5 mmoles of 2,4-dinitrophenylhydrazine was dissolved in 1 liter of 1.2 N HCI by warming the solution at 4050°C. (This must be freshly prepared every two weeks because it slowly decomposes.) Carbonyl-free ethanol. To 500 ml of reagent-grade ethanol was added about 500 mg of 2,4-dinitrophenylhydrazine and a few drops of concentrated HCl. After refluxing for more than 10 hr, the ethanol was carefully distilled, and was stored in a nitrogen atmosphere in the dark. Preparation and purification of carbonyl compounds. Commercial formaldehyde and acetaldehyde were redistilled into water after the addition of a few drops of concent,rated HCI. Other normal aliphatic aldehydes (C, to C,) were freshly prepared by oxidation of the corresponding alcohol with perchromate (5). These aldehydes were stored in dilute aqueous solution in a flask with a tight stopper in a nitrogen atmosphere in the dark. The purity of aldehyde was determined by a gravimetric 2,4-dinitrophenylhydrazine method. Benzaldehyde was purified by redistillation, and vanillin and benzophenone were used without further purification. Preparation and identification of hyd~axones of carbonyl compounds. The hydrazone of each compound was prepared in the usual way (6) and was recrystallized from benzene after desiccation over sulfuric acid. The melting point. of the compound agreed with the value in the literature. In addition, it gave a single spot on thin-layer chromatography by using ligroin/isopropyl ether (I : 1) as developing solvent. Preparation and assay of aldehyde dehydrogenase. Aldehyde dehydrogenase (EC 1.2.1.3) was prepared by Racker’s method (7) wit.h some modifications. A fresh chicken liver (40 gm) was homogenized with 3 vol of ice-cold acetone in a Waring Blendor and the mixture was poured into 8 vol of acetone. It was immediately centrifuged for 30 min at 10,000 rpm. The precipitate obtained was dried as quickly as possible over sulfuric acid in a vacuum desiccator. It was suspended (7 gm of wet cells/ 35 ml) in 20 mM Tris-HCI buffer, pH 7.5, and the mixture, after stirring for 30 min at room temperature, was centrifuged for 20 min at 1,000 rpm. The supernatant was fractionated by ammonium sulfate and the fraction between 30 urn1 60% saturation collected. The preripitatr was dis:soivcd
448
NAKAO
ARIGA
in 20 mM Tris-HCl buffer, pH 7.5, and dialyzed against the same buffer for 16 hr. The solution was centrifuged again and the supernatant used as the enzyme. The standard assay mixture contained the following components in a final volume of 4 ml: 0.6 pmole of aldehyde, 0.3 pmole of NAD, 40 pmoles of Tris-HCl buffer, pH 7.5; and the enzyme. The reaction was carried out for 30 min at 30” unless otherwise indicated. The reaction was terminated by addition of trichloroacetic acid. After removal of protein by centrifugation, the filtrate was neutralized with KOH and an aliquot of the solution was used for determination of aldehyde. METHODS
Carbonyl compounds for the determination are classified into four groups : Group 1. The compounds in this group react with the hydrazine rapidly and quantitatively under the conditions employed and the hydrazones formed are scarcely dissociable. Such compounds as keto acids, aliphatic normal aldehydes of C, to C9, vanillin, and furfural belong to this group. Group 2. The compounds in this group react with the hydrazine rapidly but the reaction does not go to completion under the conditions employed because the hydrazones are easily dissociable. Compounds such as aliphatic normal aldehydes of C, to C, and acetone belong to this group. Group 3. The compounds in group 3, such as benzaldehyde, react. fairly rapidly. The hydrazones are scarcely dissociable and are slightly soluble in alkaline solution. Group 4. The compounds in group 4, such as benzophenone, react slowly. The hydrazones are slightly soluble in alkaline solution. Method I This method is for the carbonyl compounds of groups 1 and 2. To 2 ml of sample solution in 70 vol ‘$J ethanol is added 1 ml of the hydrazine reagent. After reaction for 30 min at 30”, 7 ml of NaOH (18.4 gm/liter) is added. After 2 min, the red color produced is measured at a specific wavelength (absorption maximum) for the compound to be determined. A control measurement is made with 2 ml of 70% ethanol in place of the sample solution for each experiment. The amount of carbonyl compound (x pmole) in the sample is obtained from the following equation: A - A’ = b(k - r&r
0) A and A’ are t,he values of absorbances for the sample and for the control measurement, respectively. Ic represents the specific absorption coefficient
IETZRMIKATION
OF
CARBOiiYL
COMPOUNDS
449
for 1 pmole of the hydraeone. Although d indicates the specific absorption coefficient for 1 pmole of the hydrazine, it somewhat fluctuates slightly depending on the conditions under which the experiment is carried out and on the manner of addition of the hydrazine reagent and NaOH solution. Therefore, all the experiments, including the control measurement., should he carried out under strictly defined conditions. b represents the specific constant’ for each compound showing the degree of formation of the hydrazone derivative in a definite range of concentration under the above conditions (4). For normal aliphatic aldehydes of C5 to C, among the compounds in group 1, the following modification was made because of poor solubilities of their hydrazones in 145% ethanol: To 2 ml of sample solution (S07,S ethanol solution) is added 1 ml of the hydrazine reagent. After the reaction is carried out as described above, 4 ml of 90% ethanol and 3 ml of NaOH 142.8 gm/liter) are added to it. The red color produced is measured spectrophotometrically as described above. At this step, the final concentration of NaOH is 0.2 N and that of ethanol is about 50%. Although the value of b for such compounds as the higher aldehydes, vanillin, and furfural was actually 1, it was less than 1 for the lower aldehydes because of higher dissociabilities of their hydrazones. Accordingly this situation results in the restriction of the effective range of the concentration for the determination depending upon the kind of aldehyde. The experimental evidence for the above restriction and the det’ermination method for b has been described (4). The values k and b, effective ranges of concentration for the determination, wavelengths at which measurement is performed, and final concentration of ethanol for each aldehyde are listed in Table 1. Method
II
This method is for the carbonyl compounds in groups 3 and 4. To 2 ml of sample solution in 70 vol 5% ethanol is added 1 ml of the hydrazine reagent. After reaction for 45 min at 70” in a stoppered vessel, 10 ml of benzene and 7 ml of NaOH (18.4 mg/liter) are added with gentle shaking. After vigorous shaking, a benzene layer containing the hydrazone is separated and absorbance of the benzene solution is measured, A control measurement is made similary with 2 ml of 70% ethanol in place of the sample solution. Determination of the value of b also is carried out with t,he benzene extract obtained similary with the hydrazone. Equation 1 holds also in this case. Although benzaldehyde reacts with the hydrazine completely, benzophenone reacts with it only partially. The constants and conditions for benzaldehyde and benzophenone are listed in Table 2.
450
NAKAO
Constants
k
b
1.38 1.92 1.57 2.11 2.36 2.37 2.68 2.74 2.39 3.23 2.68
0.34 0.57 0.63 0.76 0.84 1 1 1 1 1 1
Compound
a Final b Final
concentration concentratjion
TABLE 1 for t.he Determination
and Conditions
FormaldehydeAcetaldehydea Propionaldehydea Butyraldehyde’ Valeraldehydeb Caproaldehydd Enanthaldehydeb Capryl aldehydeb Pelargonaldehydeb VanillirY Furfural”
ARIGA
of ethanol, of ethanol, RESULTS
by Method
Wavelength, w
I
Effective range of concn., pmole/lO ml
430 427 430 432 432 432 432 432 432 485 475
0.18-O. 74 0.13-2.08 0.14-2.22 0.09-1.47 0.16-0.73 0.12-0.96 0.14-1.08 0.34-0.69 0.18-0.74 0.12-1.44 0.12-0.76
14 vol %. 50 vol %. AND
DISCUSSION
Determination of aldehydes in pure solution. Table 3 shows the results of the determination for propionaldehyde, valeraldehyde, vanillin, and benzaldehyde, as examples of the determination for the carbonyl compounds by Methods I and II. As can be seen from the table, excellent recoveries were obtained. Similarly the determination of the others in the “Methods” was carried out and recoveries compounds described under from 98.5 to 1027’0 were obtained at the range of concentration shown in Tables 1 and 2. Application of the methods to assay of aldehyde dehydrogenase. Figure 1 shows NAD dependence of the enzyme when assayed using propionaldehyde as substrate. As can be seen from the figure, the activity was evidently, though not completely, depressed by omission of NAD from the complete assay system. Figure 2 shows the reactivity of the enzyme with various kinds of aldehydes at their various concentrations. Unlike the enzyme from beef Constants
and Conditions
TABLE 2 for the Determination
by Method
Wavelength, Compound Benzaldehyde Benzophenone Final
concentration
k
b
mp
3.35 2.83
1 0.065
380 385
of ethanol,
14 ~01 76.
II
Effective range of concn., pmole/lO ml 0.171.70 1.05-10.5
DETERMINATION
OF
Propionaldehyde
Valeraldehyde
Vanillin
Benzaldehyde
a Final concentration case of valeraldehyde.
rmole added
Absorbance measnred (A)a
2.22 1.11 0.560 0.280 0.140 0.725 0.633 0.319 0.160 1.44 0.960 0.480 0.240 0.120 1.70 1.36 0.680 0.340 0.170
2.6'2 1.60 1.12 0.860 0.740 1.87 1.70 1.12 0.810 4.86 3.44 1.84 1.33 0.979
of ethanol
451
COMPOUNDS
TABLE 3 of Some Aldehydes
Determination
Compound
CARBONYL
Recovery, c< /0
Method
99.1 98.2
I
101 101 109
99.2 99.2 99.3 96.3 99.5 98.5 99.0 98.5 99.0 97.1 !19.6 98.6 103
I
I
II
101
was
14 vol
y0 except
for
the use of 50 vol
4zio.75L ’y-J ‘j% % 2%_ r,
T0 in t,he
I
+NAD
050-
% 2 % 0 z 3 i= 0 Q
i---
-NAD
0.25 0.25-
O
IO
TIME
*
-
I 20
30
(min)
FIG. 1. NAD dependence of aldehyde dehydrogenase. Reaction mixture &s indicated in “Reagents and Materials” except for use of propionaldehyde as substrate. Amount of enzyme 20 mg as protein. Reaction carried out in presence and absence of NAD for indicated periods.
452
NAKAO
5 F 2
o1
ARIGA
I
0.4
I
0.8
I
1.2
CONCENTRATION OF ALDEHYDE ( pmole/ml 1
FIG. 2. Substrate
specificity of aldehyde dehydrogenase: (0) propionaldehyde, (0) butyraldehyde. Reaction mixture and conditions as indicated in “Reagents and Materials” except for use of indicated aldehydes as substrate at indicated concentrations. Activity plotted by subtracting control value, obtained by incubating mixture without substrate, from value with the complete system.
(A) acetaldehyde,
liver (7)) in the case of which acetaldehyde showed the highest activity, propionaldehyde exhibited highest activity and formaldehyde no detectable activity. This result was compatible with the one obtained by the conventional spectrophotometric method based on NADH increase. From these results, the methods presented were proved useful in the determination of small amounts of carbony compounds in bioIogica1 materials and, accordingly, in the assay of enzymes that catalyze the formation and decrease of carbonyl compounds. The methods are expected to be applicable to the determination of other carbonyl compounds in more broad categories. However, the present methods were found inapplicable for carbonyl compounds, the hydrazones of which are extremely labile in alkaline solution, such as cinnamic aldehyde and fenchone. SUMMARY
Two 2,4-dinitrophenylhydrazine methods are presented for the determination of small amounts of carbonyl compounds when present in biological materials as a single carbonyl compound. When the hydrazones of the compounds are soluble in ethanolic alkaline solution, a direct method
DETERMINATION
OF
CARBONYL
COMPOUNDS
453
is carried out omitting an extraction procedure with organic solvent. On the other hand, the extraction procedure is used when the hydrazones of the compounds are scarcely soluble in ethanolic alkaline solution. The present methods are demonstrated as being applicable to the determination of 13 compounds so far tested with good recoveries. Furthermore, they were successfully applied to the assay of aldehyde dehydrogenase. ACKNOWLEDGMENTS The author expresses his deep gratitude to Professor H. Katsuki, Department of Chemistry, Faculty of Science, Kyoto University, for valuable advice and continuous encouragement during the course of this work. He also thanks Miss Tsuya Yoshida for her encouragement. REFERENCES 1. FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 2. KATSUKI, H., KAWANO, C., YOSHIDA, T., KANAYUKI, H., AND TANAKA, S., Anal. Biochem. 2, 433 (1961). 3. KATSUKI, H., YOSHIDA, T., TANEGASHIMA, C., AND TANAKA, S., Anal. Biochem. 43,349 (1971). 4. ARIGA, N., TSUDA, M., AND KATSUKI, H., J. Chem. Sot. Jup. 88, 362 (1967). 5. HURD, C. D., MEINERT, R. N., AND SPENCE, L. U., J. Amer. Chem. Sot. 52, 1138 (1930). 6. MEISTER, A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 3, p. 404. Academic Press, New York, 1957. 7. RACKER, E., .I. Biol. Chem. 177, 883 (1949).
Methods
BIOCHEMISTRY
for
43, 446-453
(1971)
Determination
of
2,4-Dinitrophenylhydrazine to the Assay
of Chemistry,
Compounds
and
Application
of Aldehyde NAKAO
Department
Carbonyl Their
by
Dehydrogenase
ARIGA
Faculty of General Gif u, Japan
Education,
Gifu
University,
Received January 20, 1971
The determination of small amounts of carbonyl compounds in biological materials is of great importance for biochemical studies. Although many methods have been reported, the 2,4-dinitrophenylhydrazine method has been used most widely. For the determination of keto acids, the method of Friedemann and Haugen is well known (1). According to their method, the 2,4-dinitrophenylhydrazone formed by reaction with keto acid is extracted with an organic solvent from a water solution, retransferred into carbonate solution for separation from the unreacted reagent, and determined spectrophotometrically after addition of alkali. For a special sample containing a single keto acid as carbonyl compound, they proposed a direct method in which the determination was carried out omitting the extraction procedure. However, it was pointed out by Katsuki et al. that spectrophotometric measurement must be carried out under specific conditions because of the distinct molecular absorption spectra of the two geometric isomers of the hydrazone (2). From a consideration of this point, Katsuki et al. devised an improved direct method for determination of keto acids, and found that it gives satisfactory results (3). Unlike keto acids, the hydrazone of aldehyde or ketone, in general, gives one isomer under the determination conditions and consequently special consideration as to the wavelength for the determination is unnecessary. However, during studies on the determination of carbonyl compounds, the author with his collaborators pointed out the necessity for use of a special coefficient of hydrazone formation for the determination of some compounds because they form the hydrazones nonquantitatively but to a limited extent under the conditions commonly used (4). Furthermore, it was pointed out that a correction due to the coloration of the 446
DETERMINATION
OF
CARBONYL
COMPOUNDS
447
hydrazones of the carbonyl compounds present as impurities in alcohol used as solvent is necessary for the determination (4). The present paper deals with two methods devised for the determination of small amounts of aldehydes and ketones and their application to the assay of aldehyde dehydrogenase. REAGENTS
AND
MATERIALS
2,-J-Dinitrophenylhydrazine reagent. 2.5 mmoles of 2,4-dinitrophenylhydrazine was dissolved in 1 liter of 1.2 N HCI by warming the solution at 4050°C. (This must be freshly prepared every two weeks because it slowly decomposes.) Carbonyl-free ethanol. To 500 ml of reagent-grade ethanol was added about 500 mg of 2,4-dinitrophenylhydrazine and a few drops of concentrated HCl. After refluxing for more than 10 hr, the ethanol was carefully distilled, and was stored in a nitrogen atmosphere in the dark. Preparation and purification of carbonyl compounds. Commercial formaldehyde and acetaldehyde were redistilled into water after the addition of a few drops of concent,rated HCI. Other normal aliphatic aldehydes (C, to C,) were freshly prepared by oxidation of the corresponding alcohol with perchromate (5). These aldehydes were stored in dilute aqueous solution in a flask with a tight stopper in a nitrogen atmosphere in the dark. The purity of aldehyde was determined by a gravimetric 2,4-dinitrophenylhydrazine method. Benzaldehyde was purified by redistillation, and vanillin and benzophenone were used without further purification. Preparation and identification of hyd~axones of carbonyl compounds. The hydrazone of each compound was prepared in the usual way (6) and was recrystallized from benzene after desiccation over sulfuric acid. The melting point. of the compound agreed with the value in the literature. In addition, it gave a single spot on thin-layer chromatography by using ligroin/isopropyl ether (I : 1) as developing solvent. Preparation and assay of aldehyde dehydrogenase. Aldehyde dehydrogenase (EC 1.2.1.3) was prepared by Racker’s method (7) wit.h some modifications. A fresh chicken liver (40 gm) was homogenized with 3 vol of ice-cold acetone in a Waring Blendor and the mixture was poured into 8 vol of acetone. It was immediately centrifuged for 30 min at 10,000 rpm. The precipitate obtained was dried as quickly as possible over sulfuric acid in a vacuum desiccator. It was suspended (7 gm of wet cells/ 35 ml) in 20 mM Tris-HCI buffer, pH 7.5, and the mixture, after stirring for 30 min at room temperature, was centrifuged for 20 min at 1,000 rpm. The supernatant was fractionated by ammonium sulfate and the fraction between 30 urn1 60% saturation collected. The preripitatr was dis:soivcd
448
NAKAO
ARIGA
in 20 mM Tris-HCl buffer, pH 7.5, and dialyzed against the same buffer for 16 hr. The solution was centrifuged again and the supernatant used as the enzyme. The standard assay mixture contained the following components in a final volume of 4 ml: 0.6 pmole of aldehyde, 0.3 pmole of NAD, 40 pmoles of Tris-HCl buffer, pH 7.5; and the enzyme. The reaction was carried out for 30 min at 30” unless otherwise indicated. The reaction was terminated by addition of trichloroacetic acid. After removal of protein by centrifugation, the filtrate was neutralized with KOH and an aliquot of the solution was used for determination of aldehyde. METHODS
Carbonyl compounds for the determination are classified into four groups : Group 1. The compounds in this group react with the hydrazine rapidly and quantitatively under the conditions employed and the hydrazones formed are scarcely dissociable. Such compounds as keto acids, aliphatic normal aldehydes of C, to C9, vanillin, and furfural belong to this group. Group 2. The compounds in this group react with the hydrazine rapidly but the reaction does not go to completion under the conditions employed because the hydrazones are easily dissociable. Compounds such as aliphatic normal aldehydes of C, to C, and acetone belong to this group. Group 3. The compounds in group 3, such as benzaldehyde, react. fairly rapidly. The hydrazones are scarcely dissociable and are slightly soluble in alkaline solution. Group 4. The compounds in group 4, such as benzophenone, react slowly. The hydrazones are slightly soluble in alkaline solution. Method I This method is for the carbonyl compounds of groups 1 and 2. To 2 ml of sample solution in 70 vol ‘$J ethanol is added 1 ml of the hydrazine reagent. After reaction for 30 min at 30”, 7 ml of NaOH (18.4 gm/liter) is added. After 2 min, the red color produced is measured at a specific wavelength (absorption maximum) for the compound to be determined. A control measurement is made with 2 ml of 70% ethanol in place of the sample solution for each experiment. The amount of carbonyl compound (x pmole) in the sample is obtained from the following equation: A - A’ = b(k - r&r
0) A and A’ are t,he values of absorbances for the sample and for the control measurement, respectively. Ic represents the specific absorption coefficient
IETZRMIKATION
OF
CARBOiiYL
COMPOUNDS
449
for 1 pmole of the hydraeone. Although d indicates the specific absorption coefficient for 1 pmole of the hydrazine, it somewhat fluctuates slightly depending on the conditions under which the experiment is carried out and on the manner of addition of the hydrazine reagent and NaOH solution. Therefore, all the experiments, including the control measurement., should he carried out under strictly defined conditions. b represents the specific constant’ for each compound showing the degree of formation of the hydrazone derivative in a definite range of concentration under the above conditions (4). For normal aliphatic aldehydes of C5 to C, among the compounds in group 1, the following modification was made because of poor solubilities of their hydrazones in 145% ethanol: To 2 ml of sample solution (S07,S ethanol solution) is added 1 ml of the hydrazine reagent. After the reaction is carried out as described above, 4 ml of 90% ethanol and 3 ml of NaOH 142.8 gm/liter) are added to it. The red color produced is measured spectrophotometrically as described above. At this step, the final concentration of NaOH is 0.2 N and that of ethanol is about 50%. Although the value of b for such compounds as the higher aldehydes, vanillin, and furfural was actually 1, it was less than 1 for the lower aldehydes because of higher dissociabilities of their hydrazones. Accordingly this situation results in the restriction of the effective range of the concentration for the determination depending upon the kind of aldehyde. The experimental evidence for the above restriction and the det’ermination method for b has been described (4). The values k and b, effective ranges of concentration for the determination, wavelengths at which measurement is performed, and final concentration of ethanol for each aldehyde are listed in Table 1. Method
II
This method is for the carbonyl compounds in groups 3 and 4. To 2 ml of sample solution in 70 vol 5% ethanol is added 1 ml of the hydrazine reagent. After reaction for 45 min at 70” in a stoppered vessel, 10 ml of benzene and 7 ml of NaOH (18.4 mg/liter) are added with gentle shaking. After vigorous shaking, a benzene layer containing the hydrazone is separated and absorbance of the benzene solution is measured, A control measurement is made similary with 2 ml of 70% ethanol in place of the sample solution. Determination of the value of b also is carried out with t,he benzene extract obtained similary with the hydrazone. Equation 1 holds also in this case. Although benzaldehyde reacts with the hydrazine completely, benzophenone reacts with it only partially. The constants and conditions for benzaldehyde and benzophenone are listed in Table 2.
450
NAKAO
Constants
k
b
1.38 1.92 1.57 2.11 2.36 2.37 2.68 2.74 2.39 3.23 2.68
0.34 0.57 0.63 0.76 0.84 1 1 1 1 1 1
Compound
a Final b Final
concentration concentratjion
TABLE 1 for t.he Determination
and Conditions
FormaldehydeAcetaldehydea Propionaldehydea Butyraldehyde’ Valeraldehydeb Caproaldehydd Enanthaldehydeb Capryl aldehydeb Pelargonaldehydeb VanillirY Furfural”
ARIGA
of ethanol, of ethanol, RESULTS
by Method
Wavelength, w
I
Effective range of concn., pmole/lO ml
430 427 430 432 432 432 432 432 432 485 475
0.18-O. 74 0.13-2.08 0.14-2.22 0.09-1.47 0.16-0.73 0.12-0.96 0.14-1.08 0.34-0.69 0.18-0.74 0.12-1.44 0.12-0.76
14 vol %. 50 vol %. AND
DISCUSSION
Determination of aldehydes in pure solution. Table 3 shows the results of the determination for propionaldehyde, valeraldehyde, vanillin, and benzaldehyde, as examples of the determination for the carbonyl compounds by Methods I and II. As can be seen from the table, excellent recoveries were obtained. Similarly the determination of the others in the “Methods” was carried out and recoveries compounds described under from 98.5 to 1027’0 were obtained at the range of concentration shown in Tables 1 and 2. Application of the methods to assay of aldehyde dehydrogenase. Figure 1 shows NAD dependence of the enzyme when assayed using propionaldehyde as substrate. As can be seen from the figure, the activity was evidently, though not completely, depressed by omission of NAD from the complete assay system. Figure 2 shows the reactivity of the enzyme with various kinds of aldehydes at their various concentrations. Unlike the enzyme from beef Constants
and Conditions
TABLE 2 for the Determination
by Method
Wavelength, Compound Benzaldehyde Benzophenone Final
concentration
k
b
mp
3.35 2.83
1 0.065
380 385
of ethanol,
14 ~01 76.
II
Effective range of concn., pmole/lO ml 0.171.70 1.05-10.5
DETERMINATION
OF
Propionaldehyde
Valeraldehyde
Vanillin
Benzaldehyde
a Final concentration case of valeraldehyde.
rmole added
Absorbance measnred (A)a
2.22 1.11 0.560 0.280 0.140 0.725 0.633 0.319 0.160 1.44 0.960 0.480 0.240 0.120 1.70 1.36 0.680 0.340 0.170
2.6'2 1.60 1.12 0.860 0.740 1.87 1.70 1.12 0.810 4.86 3.44 1.84 1.33 0.979
of ethanol
451
COMPOUNDS
TABLE 3 of Some Aldehydes
Determination
Compound
CARBONYL
Recovery, c< /0
Method
99.1 98.2
I
101 101 109
99.2 99.2 99.3 96.3 99.5 98.5 99.0 98.5 99.0 97.1 !19.6 98.6 103
I
I
II
101
was
14 vol
y0 except
for
the use of 50 vol
4zio.75L ’y-J ‘j% % 2%_ r,
T0 in t,he
I
+NAD
050-
% 2 % 0 z 3 i= 0 Q
i---
-NAD
0.25 0.25-
O
IO
TIME
*
-
I 20
30
(min)
FIG. 1. NAD dependence of aldehyde dehydrogenase. Reaction mixture &s indicated in “Reagents and Materials” except for use of propionaldehyde as substrate. Amount of enzyme 20 mg as protein. Reaction carried out in presence and absence of NAD for indicated periods.
452
NAKAO
5 F 2
o1
ARIGA
I
0.4
I
0.8
I
1.2
CONCENTRATION OF ALDEHYDE ( pmole/ml 1
FIG. 2. Substrate
specificity of aldehyde dehydrogenase: (0) propionaldehyde, (0) butyraldehyde. Reaction mixture and conditions as indicated in “Reagents and Materials” except for use of indicated aldehydes as substrate at indicated concentrations. Activity plotted by subtracting control value, obtained by incubating mixture without substrate, from value with the complete system.
(A) acetaldehyde,
liver (7)) in the case of which acetaldehyde showed the highest activity, propionaldehyde exhibited highest activity and formaldehyde no detectable activity. This result was compatible with the one obtained by the conventional spectrophotometric method based on NADH increase. From these results, the methods presented were proved useful in the determination of small amounts of carbony compounds in bioIogica1 materials and, accordingly, in the assay of enzymes that catalyze the formation and decrease of carbonyl compounds. The methods are expected to be applicable to the determination of other carbonyl compounds in more broad categories. However, the present methods were found inapplicable for carbonyl compounds, the hydrazones of which are extremely labile in alkaline solution, such as cinnamic aldehyde and fenchone. SUMMARY
Two 2,4-dinitrophenylhydrazine methods are presented for the determination of small amounts of carbonyl compounds when present in biological materials as a single carbonyl compound. When the hydrazones of the compounds are soluble in ethanolic alkaline solution, a direct method
DETERMINATION
OF
CARBONYL
COMPOUNDS
453
is carried out omitting an extraction procedure with organic solvent. On the other hand, the extraction procedure is used when the hydrazones of the compounds are scarcely soluble in ethanolic alkaline solution. The present methods are demonstrated as being applicable to the determination of 13 compounds so far tested with good recoveries. Furthermore, they were successfully applied to the assay of aldehyde dehydrogenase. ACKNOWLEDGMENTS The author expresses his deep gratitude to Professor H. Katsuki, Department of Chemistry, Faculty of Science, Kyoto University, for valuable advice and continuous encouragement during the course of this work. He also thanks Miss Tsuya Yoshida for her encouragement. REFERENCES 1. FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 2. KATSUKI, H., KAWANO, C., YOSHIDA, T., KANAYUKI, H., AND TANAKA, S., Anal. Biochem. 2, 433 (1961). 3. KATSUKI, H., YOSHIDA, T., TANEGASHIMA, C., AND TANAKA, S., Anal. Biochem. 43,349 (1971). 4. ARIGA, N., TSUDA, M., AND KATSUKI, H., J. Chem. Sot. Jup. 88, 362 (1967). 5. HURD, C. D., MEINERT, R. N., AND SPENCE, L. U., J. Amer. Chem. Sot. 52, 1138 (1930). 6. MEISTER, A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 3, p. 404. Academic Press, New York, 1957. 7. RACKER, E., .I. Biol. Chem. 177, 883 (1949).