ANALYTICAL
BIOCHEMISTRY
66, 365-371 (1975)
An Investigation of the Assay Using Trinitrobenzenesulphonic
of Dopamine Acid
ALAN CHARTERIS AND ROBERTJOHN Department P.O.
of Biochemistry, Box 78, Cardiff
CFl
University College, lXL, U.K.
Received September 30, 1974; accepted January 16, 1975 The reaction between 2,4,6-trinitrobenzene-I-sulphonic acid with 3,4-dihydroxyphenylethylamine (dopamine) to form trinitrophenyl dopamine (TNP-dopamine) is found to be complex in that the TNP-dopamine is unstable. The course of the reaction shows TNP-dopamine rising to a steady state concentration equal to about half the initial dopamine concentration within 20 min and then decomposing with a half-time of about 1 hr. Continuous extraction into benzene to separate the TNP-dopamine from TNP-DOPA, which interferes with the spectrophotometric assay, also prevents the decomposition of TNP-dopamine so that quantitative recoveries are obtained. The method has been used to follow the course of a DOPA decarboxylase-catalysed reaction satisfactorily.
A useful method for the determination of 3,4-dihydroxyphenylethylamine (dopamine) in the presence of 3,4-dihydroxy L-phenylalanine (DOPA) has been exploited by Streffer (1) in the assay of L-DOPA aromatic amino acid decarboxylase (3,4-dihydroxy-phenylalanine-carboxylyase, EC 4.11.26). After initial incubation of the enzyme with DOPA the reaction is stopped with perchloric acid which is then neutralised. The dopamine formed is treated with 2,4,6-trinitrobenzene1-sulphonic acid (TNBS) with which it reacts to form trinitrophenyl-1-dopamine (TNP-dopamine). This compound is then extracted into benzene and its concentration determined spectrophotometrically. The benzene extraction is necessary to separate the TNP-dopamine from TNP-DOPA which would otherwise interfere since it also absorbs in the same region. A slight alteration has been made to this procedure by Sherald et al. (2) in which KCN is used to stop the enzymic reaction rather than perchloric acid. When used in this laboratory this method gives extinction values that are in good agreement with those found by Sherald et al. (2) but about 50% lower than would be expected from the extinction coefficient given by Streffer (1). Since this extinction coefficient is very similar to those quoted for a large number of TNP-amino acids (3) it seemed likely that the discrepancy was not due to an incorrect value for the extinction 365 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
366
CHARTERIS
AND
JOHN
coefficient but rather to a poor yield of the TNP derivative. an account of investigations carried out into the method. METHODS
This paper is
AND MATERIALS
Reagents. TNBS, dopamine and iproniazid phosphate were bought from Sigma (London) Chemical Co., London SW6, U.K. DOPA and pyridoxal-5’-phosphate were bought from Koch-Light Laboratories Ltd., Colnbrook, Bucks., U.K. All other reagents were bought from British Drug Houses, Poole, Dorset, U.K. Spectrophotometric determinations. Measurements of TNP-dopamine concentration were carried out using a Cecil CE 272 spectrophotometer using e340= 1.24 X lo4 M-’ (1). DOPA decarboxylase. The enzyme was isolated from hog kidney and purified by heat treatment and (NH4)&S04 fractionation according to Lancaster and Sourkes (4). RESULTS Progress of Reaction
Between TNBS
and Dopamine
Solutions, each 1.25 ml, containing 7.2 X 1Op5 M dopamine (9.0 X 1Op8 moles in 1.25 ml), 2.4 X 1O-4 M DOPA, 0.1 mM iproniazid phosphate, 1.6 X IO5 M pyridoxal-5’-phosphate, 3.4 mM TNBS and 2.0 mM KCN in 0.1~ sodium phosphate buffer, pH 7.3, were maintained at 42°C for increasing periods of time and then extracted with 1.5 ml of benzene and the TNP dopamine concentration determined. The results, after subtraction of control values obtained using solutions from which dopamine was omitted, show (Fig. 1) a rise to a maximum level of 3.9 X 1O-5 M (5.1 X lo-* moles in 1.5 ml of benzene) over a period of about 20 min followed by a continuous fall. These observations show that there are two consecutive reactions taking place whereby the formation of TNPdopamine is followed by its breakdown. The maximum yield obtainable under these conditions is 57% at 20 min. The results are a reasonably good fit to a scheme involving an initial second-order reaction with rate constant kl = 17.7 M-’ min-’ followed by a first order breakdown with a rate constant k, = 0.025 mm-‘. Considering the course of the reaction it is not surprising to find that a calibration curve constructed according to Sherald et al. (2) does not give values in accord with the extinction coefficient. The conditions used by Sherald et al. (2), which are essentially those described above, were used to construct a standard curve which is shown in Fig. 2(A). Continuous Extraction of TNP-Dopamine with Benzene It was observed that the extinction of TNP-dopamine in benzene remained constant over a period of at least 90 min, and it seemed proba-
ASSAY
I 0
OF
367
DOPAMINE
I LO
I 80 TIME
1 120
IminI
1. Course of the reaction of TNBS with dopamine. The points are experimental values obtained as explained in the text. The solid line was obtained for the concentration of the intermediate in a scheme A + B + C, in which A represents dopamine and B, TNP dopamine. The equation used was FIG.
The first step was taken to be pseudo-first order (because of the large excess of TNBS with rate constant k, = 0.06 min-’ corresponding to a second-order constant k = 17.7 M-‘min-‘. The second reaction was taken to be first order with k, = 0.025 min-I.
ble therefore that continuous extraction into benzene would prevent the decomposition of the compound and provide a stoichiometric assay. A solution (37.5 ml) containing the concentrations of reactants described earlier was vigorously stirred in the presence of 45 ml of benzene, samples of the benzene layer removed at 5-min intervals and the concentration of TNP-dopamine determined. The results are shown in Fig. 3 from which it is clear that the dopamine is converted quantitatively to its TNP conjugate. These results and subsequent routine assays were achieved using individual, tightly capped, 20-ml scintillation vials which had been modified by drawing out the bases to give a round bottom that fitted the clips in the rack of a Gallenkamp shaking reaction incubator. Each vial contained 1.25 ml of the solution of reactants described above and was shaken vigorously at 42°C for 1 hr. Figure 2(B) shows the calibration curve so obtained.
368
CHARTERIS
AND JOHN
0.9-
0.8 0.7,E 2 0
0.6 -
z 3 G rg
0,5-
$
0,3-
k
04-
0.2 0.1 -
n moles
DOPAMINE
PER
0.25 ml
REACTION
FIG. 2. Calibration curves obtained using (A) 20-min incubation followed by extraction with benzene and (B) continuous extraction with benzene. For experimental details see text.
0
50
100 TIME
150
(mini
FIG. 3. Course of the reaction of TNBS with dopamine using continuous benzene extraction. For experimental details see text.
369
ASSAY OF DOPAMINE
Dependence of Reaction Rate on pH
The reaction between dopamine and TNBS would be expected to be a nucleophilic substitution of the a-amino group of dopamine at the sulphonic acid-bearing carbon of the TNBS. The rate of the reaction should be pH dependent since only the un-ionised form of the amino group should be reactive. The progress of the reaction was followed at several pH values using the concentrations described above and individual capped vials with continuous extraction into benzene. A plot of apparent first-order rate constant against pH is shown in Fig. 4 in which the points are experimental and the solid line obtained theoretically, assuming the pK of the amino group to be 8.1 and the maximum value of the rate constant to be 0.42 min-‘. Although the reaction rate increases considerably with pH, at higher pH values the yield of TNP-dopamine falls short of that expected for complete conversion. This is probably due to the instability of dopamine at high pH.
I 8.0
I
PH
FIG. 4. pH dependence of reaction of TNBS with dopamine. The points are experimental and were obtained using reactant concentrations described in the text. For points below pH 7.5, 0.1 M sodium phosphate buffers were used and for points above pH 7.5, 0.05 M sodium barbitone corrected with HCI was used. The solid line is theoretical constructed according to k,, = k,,,/( l@pK--pH)+ 1) in which k,, = 0.42 mine1 and pK = 8.1.
370
CHARTERS
AND JOHN
V 0 Time
I
I
30
60 Cminl
5. Course of DOPA decarboxylase reaction followed using continuous benzene extraction. For details see text. Results are expressed as concentration of dopamine in the enzyme assay solution. FIG.
Assay of DOPA Decarboxylase
The progress of dopamine formation in the presence of a preparation of DOPA decarboxylase was followed. The reaction was started by adding 0.05 ml of a suitably diluted enzyme preparation to scintillation vials containing 0.20 ml of a solution buffered at pH 7.0 with 0.1 M sodium phosphate. The final concentrations in the incubation mixture were: DOPA, 1.2 mu; iproniazid phosphate, 1 mu, and pyridoxal phosphate, 0.08 mu. The solutions were incubated for various times at 37°C and the reaction stopped by heating to 100°C for 1 min. Benzene (1.5 ml) and TNBS (1 ml, 4.3 mu) were added to each vial and a second reaction to form TNP-dopamine was carried out at 42°C for 1 hr with continuous shaking. The TNP-dopamine concentration in the benzene layer was measured as described above. The results are shown in Fig. 5. DISCUSSION
The results presented in this paper show that the assay of dopamine using TNBS is complicated by the instability of the TNP-dopamine formed. Despite this instability, Sherald (2) has found a linear correlation between dopamine concentration and the eventual extinction value obtained, and our results confirm this observation. Such behaviour is entirely consistent with a reaction scheme in which there are two consecutive steps. Thus, so long as the rate constants governing the two steps
371
ASSAY OF DOPAMINE
are not altered, the maximum level of TNP-dopamine will always be reached at the same time and will always represent the same fraction of the initial dopamine used. However, any method that relies on determining concentrations during a short steady state is less satisfactory than one in which a stoichiometric yield of stable product is obtained since the time of incubation is much more critical. This is because any change in the reaction mixture which affects the rates of formation or breakdown of TNP-dopamine will affect the concentration of this compound present at any fixed time. For example a lowering of pH from 7.5 to 7.3 will produce a reduction in rate constant of 30% and this will lead to an estimate of dopamine concentration which will be 19% lower at pH 7.3 than at pH 7.5. If the Sherald (2) method were used to investigate the effect of pH on the DOPA decarboxylase reaction, it would be important to ensure that even small deviations in the pH of the dopamine assay solution were corrected. Such small changes in pH need not be corrected if continuous benzene extraction is used since under these conditions the reaction goes to completion and, so long as the time of incubation is long enough and the pH is low enough to prevent decomposition of dopamine, quantitative yields of TNP-dopamine are achieved. The method gives results for the assay of DOPA decarboxylase which compare well with those obtained using other methods (5). The pH dependence of the reaction is interesting in that it shows participation of a group ionising with a pK of 8.1. The pK of the dopamine amino group has been determined to be 8.9 (6), and it seems probable that it is lowered by the formation of an initial charge transfer complex in which the two aromatic rings are stacked with the sulphonate of TNBS juxtaposed to the amino group of dopamine thus decreasing the affinity of the amino group for its proton. It is also possible however that at least some of the difference in pK is due to temperature and ionic strength effects. REFERENCES 1. Streffer, C. (1967) Biochim. Biophys. Acta. 139, 193-195. 2. Shera.ld, A. F., Sparrow, J. C., and Wright, T. R. F. (1973) Anal. Biochem.
56,
300-305. 3.
Sat&e, K., Okuyama, T., Ohashi, M., and Skinoda, T. (1960) J. Biochem. Tokyo
47,
654-660. 4. Lancaster, G. A., and Sourkes, T. L. (1972) Can. J. Biochem. 50, 791-797. 5. Davis, V. E., and Awapara, J. (1960) J. Biol. Chem. 235, 124-127. 6. Tuckerman, M. M., Mayer, J. R., and Nachod, F. C. (1959) J. Amer. Chem. 92-94.
Sot.
81,
BIOCHEMISTRY
66, 365-371 (1975)
An Investigation of the Assay Using Trinitrobenzenesulphonic
of Dopamine Acid
ALAN CHARTERIS AND ROBERTJOHN Department P.O.
of Biochemistry, Box 78, Cardiff
CFl
University College, lXL, U.K.
Received September 30, 1974; accepted January 16, 1975 The reaction between 2,4,6-trinitrobenzene-I-sulphonic acid with 3,4-dihydroxyphenylethylamine (dopamine) to form trinitrophenyl dopamine (TNP-dopamine) is found to be complex in that the TNP-dopamine is unstable. The course of the reaction shows TNP-dopamine rising to a steady state concentration equal to about half the initial dopamine concentration within 20 min and then decomposing with a half-time of about 1 hr. Continuous extraction into benzene to separate the TNP-dopamine from TNP-DOPA, which interferes with the spectrophotometric assay, also prevents the decomposition of TNP-dopamine so that quantitative recoveries are obtained. The method has been used to follow the course of a DOPA decarboxylase-catalysed reaction satisfactorily.
A useful method for the determination of 3,4-dihydroxyphenylethylamine (dopamine) in the presence of 3,4-dihydroxy L-phenylalanine (DOPA) has been exploited by Streffer (1) in the assay of L-DOPA aromatic amino acid decarboxylase (3,4-dihydroxy-phenylalanine-carboxylyase, EC 4.11.26). After initial incubation of the enzyme with DOPA the reaction is stopped with perchloric acid which is then neutralised. The dopamine formed is treated with 2,4,6-trinitrobenzene1-sulphonic acid (TNBS) with which it reacts to form trinitrophenyl-1-dopamine (TNP-dopamine). This compound is then extracted into benzene and its concentration determined spectrophotometrically. The benzene extraction is necessary to separate the TNP-dopamine from TNP-DOPA which would otherwise interfere since it also absorbs in the same region. A slight alteration has been made to this procedure by Sherald et al. (2) in which KCN is used to stop the enzymic reaction rather than perchloric acid. When used in this laboratory this method gives extinction values that are in good agreement with those found by Sherald et al. (2) but about 50% lower than would be expected from the extinction coefficient given by Streffer (1). Since this extinction coefficient is very similar to those quoted for a large number of TNP-amino acids (3) it seemed likely that the discrepancy was not due to an incorrect value for the extinction 365 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
366
CHARTERIS
AND
JOHN
coefficient but rather to a poor yield of the TNP derivative. an account of investigations carried out into the method. METHODS
This paper is
AND MATERIALS
Reagents. TNBS, dopamine and iproniazid phosphate were bought from Sigma (London) Chemical Co., London SW6, U.K. DOPA and pyridoxal-5’-phosphate were bought from Koch-Light Laboratories Ltd., Colnbrook, Bucks., U.K. All other reagents were bought from British Drug Houses, Poole, Dorset, U.K. Spectrophotometric determinations. Measurements of TNP-dopamine concentration were carried out using a Cecil CE 272 spectrophotometer using e340= 1.24 X lo4 M-’ (1). DOPA decarboxylase. The enzyme was isolated from hog kidney and purified by heat treatment and (NH4)&S04 fractionation according to Lancaster and Sourkes (4). RESULTS Progress of Reaction
Between TNBS
and Dopamine
Solutions, each 1.25 ml, containing 7.2 X 1Op5 M dopamine (9.0 X 1Op8 moles in 1.25 ml), 2.4 X 1O-4 M DOPA, 0.1 mM iproniazid phosphate, 1.6 X IO5 M pyridoxal-5’-phosphate, 3.4 mM TNBS and 2.0 mM KCN in 0.1~ sodium phosphate buffer, pH 7.3, were maintained at 42°C for increasing periods of time and then extracted with 1.5 ml of benzene and the TNP dopamine concentration determined. The results, after subtraction of control values obtained using solutions from which dopamine was omitted, show (Fig. 1) a rise to a maximum level of 3.9 X 1O-5 M (5.1 X lo-* moles in 1.5 ml of benzene) over a period of about 20 min followed by a continuous fall. These observations show that there are two consecutive reactions taking place whereby the formation of TNPdopamine is followed by its breakdown. The maximum yield obtainable under these conditions is 57% at 20 min. The results are a reasonably good fit to a scheme involving an initial second-order reaction with rate constant kl = 17.7 M-’ min-’ followed by a first order breakdown with a rate constant k, = 0.025 mm-‘. Considering the course of the reaction it is not surprising to find that a calibration curve constructed according to Sherald et al. (2) does not give values in accord with the extinction coefficient. The conditions used by Sherald et al. (2), which are essentially those described above, were used to construct a standard curve which is shown in Fig. 2(A). Continuous Extraction of TNP-Dopamine with Benzene It was observed that the extinction of TNP-dopamine in benzene remained constant over a period of at least 90 min, and it seemed proba-
ASSAY
I 0
OF
367
DOPAMINE
I LO
I 80 TIME
1 120
IminI
1. Course of the reaction of TNBS with dopamine. The points are experimental values obtained as explained in the text. The solid line was obtained for the concentration of the intermediate in a scheme A + B + C, in which A represents dopamine and B, TNP dopamine. The equation used was FIG.
The first step was taken to be pseudo-first order (because of the large excess of TNBS with rate constant k, = 0.06 min-’ corresponding to a second-order constant k = 17.7 M-‘min-‘. The second reaction was taken to be first order with k, = 0.025 min-I.
ble therefore that continuous extraction into benzene would prevent the decomposition of the compound and provide a stoichiometric assay. A solution (37.5 ml) containing the concentrations of reactants described earlier was vigorously stirred in the presence of 45 ml of benzene, samples of the benzene layer removed at 5-min intervals and the concentration of TNP-dopamine determined. The results are shown in Fig. 3 from which it is clear that the dopamine is converted quantitatively to its TNP conjugate. These results and subsequent routine assays were achieved using individual, tightly capped, 20-ml scintillation vials which had been modified by drawing out the bases to give a round bottom that fitted the clips in the rack of a Gallenkamp shaking reaction incubator. Each vial contained 1.25 ml of the solution of reactants described above and was shaken vigorously at 42°C for 1 hr. Figure 2(B) shows the calibration curve so obtained.
368
CHARTERIS
AND JOHN
0.9-
0.8 0.7,E 2 0
0.6 -
z 3 G rg
0,5-
$
0,3-
k
04-
0.2 0.1 -
n moles
DOPAMINE
PER
0.25 ml
REACTION
FIG. 2. Calibration curves obtained using (A) 20-min incubation followed by extraction with benzene and (B) continuous extraction with benzene. For experimental details see text.
0
50
100 TIME
150
(mini
FIG. 3. Course of the reaction of TNBS with dopamine using continuous benzene extraction. For experimental details see text.
369
ASSAY OF DOPAMINE
Dependence of Reaction Rate on pH
The reaction between dopamine and TNBS would be expected to be a nucleophilic substitution of the a-amino group of dopamine at the sulphonic acid-bearing carbon of the TNBS. The rate of the reaction should be pH dependent since only the un-ionised form of the amino group should be reactive. The progress of the reaction was followed at several pH values using the concentrations described above and individual capped vials with continuous extraction into benzene. A plot of apparent first-order rate constant against pH is shown in Fig. 4 in which the points are experimental and the solid line obtained theoretically, assuming the pK of the amino group to be 8.1 and the maximum value of the rate constant to be 0.42 min-‘. Although the reaction rate increases considerably with pH, at higher pH values the yield of TNP-dopamine falls short of that expected for complete conversion. This is probably due to the instability of dopamine at high pH.
I 8.0
I
PH
FIG. 4. pH dependence of reaction of TNBS with dopamine. The points are experimental and were obtained using reactant concentrations described in the text. For points below pH 7.5, 0.1 M sodium phosphate buffers were used and for points above pH 7.5, 0.05 M sodium barbitone corrected with HCI was used. The solid line is theoretical constructed according to k,, = k,,,/( l@pK--pH)+ 1) in which k,, = 0.42 mine1 and pK = 8.1.
370
CHARTERS
AND JOHN
V 0 Time
I
I
30
60 Cminl
5. Course of DOPA decarboxylase reaction followed using continuous benzene extraction. For details see text. Results are expressed as concentration of dopamine in the enzyme assay solution. FIG.
Assay of DOPA Decarboxylase
The progress of dopamine formation in the presence of a preparation of DOPA decarboxylase was followed. The reaction was started by adding 0.05 ml of a suitably diluted enzyme preparation to scintillation vials containing 0.20 ml of a solution buffered at pH 7.0 with 0.1 M sodium phosphate. The final concentrations in the incubation mixture were: DOPA, 1.2 mu; iproniazid phosphate, 1 mu, and pyridoxal phosphate, 0.08 mu. The solutions were incubated for various times at 37°C and the reaction stopped by heating to 100°C for 1 min. Benzene (1.5 ml) and TNBS (1 ml, 4.3 mu) were added to each vial and a second reaction to form TNP-dopamine was carried out at 42°C for 1 hr with continuous shaking. The TNP-dopamine concentration in the benzene layer was measured as described above. The results are shown in Fig. 5. DISCUSSION
The results presented in this paper show that the assay of dopamine using TNBS is complicated by the instability of the TNP-dopamine formed. Despite this instability, Sherald (2) has found a linear correlation between dopamine concentration and the eventual extinction value obtained, and our results confirm this observation. Such behaviour is entirely consistent with a reaction scheme in which there are two consecutive steps. Thus, so long as the rate constants governing the two steps
371
ASSAY OF DOPAMINE
are not altered, the maximum level of TNP-dopamine will always be reached at the same time and will always represent the same fraction of the initial dopamine used. However, any method that relies on determining concentrations during a short steady state is less satisfactory than one in which a stoichiometric yield of stable product is obtained since the time of incubation is much more critical. This is because any change in the reaction mixture which affects the rates of formation or breakdown of TNP-dopamine will affect the concentration of this compound present at any fixed time. For example a lowering of pH from 7.5 to 7.3 will produce a reduction in rate constant of 30% and this will lead to an estimate of dopamine concentration which will be 19% lower at pH 7.3 than at pH 7.5. If the Sherald (2) method were used to investigate the effect of pH on the DOPA decarboxylase reaction, it would be important to ensure that even small deviations in the pH of the dopamine assay solution were corrected. Such small changes in pH need not be corrected if continuous benzene extraction is used since under these conditions the reaction goes to completion and, so long as the time of incubation is long enough and the pH is low enough to prevent decomposition of dopamine, quantitative yields of TNP-dopamine are achieved. The method gives results for the assay of DOPA decarboxylase which compare well with those obtained using other methods (5). The pH dependence of the reaction is interesting in that it shows participation of a group ionising with a pK of 8.1. The pK of the dopamine amino group has been determined to be 8.9 (6), and it seems probable that it is lowered by the formation of an initial charge transfer complex in which the two aromatic rings are stacked with the sulphonate of TNBS juxtaposed to the amino group of dopamine thus decreasing the affinity of the amino group for its proton. It is also possible however that at least some of the difference in pK is due to temperature and ionic strength effects. REFERENCES 1. Streffer, C. (1967) Biochim. Biophys. Acta. 139, 193-195. 2. Shera.ld, A. F., Sparrow, J. C., and Wright, T. R. F. (1973) Anal. Biochem.
56,
300-305. 3.
Sat&e, K., Okuyama, T., Ohashi, M., and Skinoda, T. (1960) J. Biochem. Tokyo
47,
654-660. 4. Lancaster, G. A., and Sourkes, T. L. (1972) Can. J. Biochem. 50, 791-797. 5. Davis, V. E., and Awapara, J. (1960) J. Biol. Chem. 235, 124-127. 6. Tuckerman, M. M., Mayer, J. R., and Nachod, F. C. (1959) J. Amer. Chem. 92-94.
Sot.
81,
