- Email: [email protected]
A microtiter plate-based assay for phosphoenolpyruvate carboxylase
ANALYTICAL
BIOCHEMISTRY
189,
95-98
(1990)
A Microtiter Plate-Based Assay for Phosphoenolpyruvate Carboxylase William Cockburn,* Garry C. Whitelam,* Stephen P. Slocombe,* and Raymond A. McKee? Botany Department, School of Biological Sciences, University of Leicester, Leicester, LEl 7RH, United Kingdom; and A.F.R.C. Food Research Institute, Colney Lane, Norwich, NR4 7UA, United Kingdom
Received
October
20,1989
A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography. 0 1990 Academic Press, Inc.
achieved by two distinct methods. Nimmo (5) describes a system in which orthophosphate released from PEP in the carboxylation reaction is visualized by precipitation as calcium phosphate. The second and most commonly utilized method relies upon the reaction between oxaloacetate and Fast Violet B, which results in the generation of a colored precipitate (6). The present work describes a sensitive, quantitative method for the assay of PEPC which is based on the Fast Violet reaction and which, because it is carried out in microtiter plates, is particularly suited to the analysis of large numbers of samples. MATERIALS
Phosphoenolpyruvate carboxylase (PEPC)l (EC 4.1.1.31) is usually assayed by coupling the reduction of oxaloacetate produced in the PEPC reaction, by means of malic dehydrogenase (EC 1.1.1.37), to the oxidation of NADH, which is monitored spectrophotometrically (1,2). An improvement in this venerable method, in which pyruvate resulting from the decomposition of oxaloacetate is reduced to lactate by lactate dehydrogenase and therefore contributes to the oxidation of NADH, has recently been suggested (3). Although precise and sensitive this assay is by no means ideal for the monitoring of column eluates or other situations in which large numbers of determinations must be made. PEPC activity has also been measured as the incorporation of radiocarbon from [14C]bicarbonate into oxaloacetate (4). Although very sensitive, and adaptable to use with relatively large numbers of samples, the method suffers from the expense of radioisotopes and the inconvenience and potential hazards of working with volatile radioactive substances. Qualitative localization of PEPC on gels has been ’ Abbreviations used: PEP, phosphoenolpyruvate;
0003-‘2697/QO
Copyright All rights
PEPC, phosphoenolpyruvate TCA, tricarboxylic acid.
$3.00
0 1990 by Academic of reproduction
Press, Inc.
in any form
reserved.
carboxylase;
AND
METHODS
Chemicals and enzymes. Phosphoenolpyruvate, phosphoenolpyruvate carboxylase, malic dehydrogenase, Fast Violet B salt, sodium diethyldithiocarbamate, polyvinylpolypyrrolidone, and Tris buffer were obtained from Sigma Chemical Co. Other chemicals were obtained from BDH Chemicals Ltd., Poole, England. Preparation of crude enzyme extract. Samples of leaf lamina weighing 1.0 g from 12-day-old maize plants were ground to a fine powder in liquid nitrogen. The frozen powder was extracted with 5.0 ml of a solution containing 50 mM Tris/HCl, 30 m&f sodium isoascorbate, 2.0 mM &sodium EDTA, and 1% polyvinylpolypyrrolidone, pH 7.2. The homogenate was centrifuged for 2 min at 12,000 rpm in a bench microfuge and the supernatant solution decanted and stored on ice until required. Aliquots of crude Size exclusion chromatography. enzyme solution (200 ~1) were separated on an LKB GlasPac G3000SW column, using 50 mM Tris/HCl, pH 7.2, containing 2.0 mM dithiothreitol as eluent at a flow rate of 0.4 ml/min. The column eluate was monitored at 280 nm. 95
96
COCKBURN
ET
Spectrophotometric assay. A solution containing 50 Tris/HCl, 10 mM KHCO,, 5.0 mM MgCl,, 2.0 mM PEP, 10 units malic dehydrogenase, 0.2 mM NADH, pH 7.8, was monitored at 340 nm. The reaction was started by the addition of enzyme to bring the final volume to 1.0 ml. Some assays were started by the addition of PEP in order to assess background rates of NADH oxidation. Microtiter plate assay. The final concentrations of reagents in the microtiter plate wells was 100 mM Tris, 40 mM NaHCO,, 10 mM MgCl,, pH 7.2 (added as 25 ~1 of a single stock solution), and 6.0 mM PEP, which was added separately (as 25 ~1 of 24 mM stock solution) to permit the use of minus-substrate controls. The reaction was started by the addition of the enzyme solution, water having been added to ensure the final volume in the well would be 100 ~1. After 10 min incubation with gentle shaking at room temperature (2O“C) 25 ~1 of a 3.0% (wt/vol) solution of Fast Violet B was added to give a final concentration of 0.6%. Following a further lomin incubation the reaction was stopped by the addition of 25 ~1 of 80% lactic acid (vol/vol) to give a final concentration of 13%. After 5.0 min the plate was read at 490 nm using an empty well as blank and the data were expressed as the difference between samples with and without PEP substrate. The color was stable for at least an overnight period. Stock solutions were stored frozen. Once thawed the PEP and Fast Violet solutions were stored on ice.
AL. 0.60
mM
RESULTS
AND
0.46
0.36 z Iii e 2 2 0.24 J
0.12
DISCUSSION
0.00 400
Assay PH. A pH of 7.2 was selected for the standard assay conditions. It was found that the use of a pH greater than 7.8 resulted in the deposition of precipitates in both plus and minus PEP wells which did not redissolve following acidification and which interfered with subsequent optical quantification. As Vidal et al. (6) indicated, mercaptoethanol, isoascorbate, and thioglycollate cause increased background values and are best avoided when possible. Order of addition of reagents. Addition of Fast Violet to the assay after a period of incubation rather than including it from the outset of the assay resulted in enhancement of the difference between plus and minus PEP assays. The reason for this is not known although it is possible that the enzyme is protected from some deleterious effect of Fast Violet by prior incubation with its substrate. The enzyme reaction and the generation of colored product continued in the presence of Fast Violet. Addition of the reagents in the order prescribed also resulted in the deposition of a finely divided precipitate which dissolved readily when the assay was terminated by acidification. Wavelength at which to read assay. Figure 1 shows a difference spectrum of plus and minus PEP replicates
I
I
I
500
600
700
Wavelength FIG. PEP
1. Optical absorbance assay solutions.
difference
(nm) spectrum
of plus
and minus
subjected to the standard assay conditions. The wavelength range between approximately 500 and 550 would be optimal in terms of both magnitude of extinction coefficient and insensitivity to minor wavelength variations. Obviously it is not essential to work within the optimum range and in fact the plate reader, which performed perfectly satisfactorily in the present work, was fitted with a 490-nm filter. Reproducibility of the assay. Analysis of six replicate samples using the standard assay conditions gave a mean value of 0.194 with a sample standard variation of 0.0098. The reproducibility of the assay is therefore satisfactory for most purposes and is certainly more than adequate for monitoring chromatographic eluates. Linearity of response. The linearity of the assay with time, using commercially obtained enzyme, is shown in Fig. 2. Differences between plus and minus PEP wells continued to increase throughout the experiment and
PHOSPHOENOLPYRUVATE
CARBOXYLASE
97
ASSAY
0.06 0.05 s 0.04 al z u 0.03 -
a
0.02 0.01
I
0
I 10
5
I 15 Time
I 20
I 25
I 30
[mid
FIG. 2. Relationship between incubation time and magnitude of difference between optical density reading at 490 nm (AA,& of plus and minus PEP replicate assays. Y = -2.45 X 10m3 (22.26 X 1Om3) + 2.03 X 10e3X (11.24 X 10m4). Determination coefficient, 99.8%.
l1 .t i
I
I 2.4
Elution
although a total incubation time of 20 min was chosen for the standard assay it is clear that incubation periods up to and beyond 30 min are practicable at room temperature. Assays were incubated at room temperature partly for convenience and partly because incubation at higher temperatures increased minus-substrate background levels. Sensitivity of the assay and response to amount of enzyme. The assay was performed with dilutions of com-
0.03 ii P -F
a
0.02
J 0
0.4 PEP carboxylase
0.8
1.2 activity
1.6 units
FIG. 3. Relationship between amounts of enzyme activity present in assay and the difference between optical density readings at 490 nm (AA,& of plus and minus PEP replicate assays. (One unit of enzyme activity catalyzes the synthesis of 1 pmol of oxaloacetate per minute at 20°C.) Y = 2.37 X 10e3 (fl.O1 X lo-‘) + 2.09 X 10m3X (k1.09 X lOma). Determination coefficient, 99.5%.
I volume
I 3.2
I .O (ml)
FIG. 4. Estimation of PEP carboxylase activity by the Fast Violet assay (difference between optical density at 490 nm of plus and minus PEP replicate assays (AA& (0) and optical density at 280 nm (A,) (-) of column eluate fractions from size-exclusion chromatography of a crude extract of leaves of &a nays.
mercial PEPC and it was found that with the incubation times specified it was possible to detect 0.07 units of the enzyme and also that there was a linear response to the amount of enzyme assayed (Fig. 3). The sensitivity observed is similar to that of the conventional spectrophotometric assay. No doubt the sensitivity of both methods could be increased were this a prime consideration. The sensitivity of the standard Fast Violet microtiter plate assay readily permits the measurement of PEPC activity in the column eluate resulting from the size-exclusion chromatography of an unconcentrated crude extract of maize leaves (Fig. 4). Partial separation of PEPC from ribulosebisphosphate carboxylase/oxygenase has been achieved and the enzyme activity coincides with one of the two, partially resolved, protein peaks. The assay is therefore fully satisfactory for the purpose for which it is likely to be most useful-that is the monitoring of chromatographic fractions during purification procedures. PEPC is a component of a number of metabolic pathways including C, and CAM photosynthesis, stomata1 mechanism, anaplerosis in the TCA cycle, and pH regulation. These varied roles impose a range of constraints and requirements on the regulatory characteristics of PEPC, and isoforms of this enzyme, which may well be pathway-specific, are increasingly being discovered
98
COCKBURN
(5,7). Detailed study of these isoforms to determine their kinetic characteristics will inevitably involve chromatographic separation. The microtiter plate assay described here is particularly useful in the monitoring of chromatographic fractions and, while offering equivalent sensitivity, is more convenient and economical than the conventional spectrophotometric assay.
ET
AL.
REFERENCES 1. Bandurski, R. S., and Greiner, 781-786. 2. Walker, D. A. (1957) Biochem. 3. Meyer,
R. C., Rustin,
(1953)
J. 67,
73-79.
P., and Wedding,
J. Biol.
R. T. (1988)
Chem.
Plant
204,
Physiol.
86,325-328. 4. Jones, R., Wilkins, colm,
A. D. B. (1978)
M. B., Coggins, Biochem. J.
5. Nimmo, G. A., Nimmo, J. 239,213-220.
ACKNOWLEDGMENT
C. M.
6. Vidal,
J., Cavalie,
J. R., Fewson,
H. G., and Wilkins,
G., and Vidal,
C. A., and Mal-
175,391-406.
P. (1976)
M. B. (1986) Plant
Sci. Lett.
Biochem. 7,265-
270. The authors are very tion of diagrams.
grateful
to Mrs.
Sue Ogden
for the prepara-
7. Cushman, and Bohnert,
J. C., Meyer, G., Michalowski, H. J. (1989) Plant Cell 1,
C. B., Schmitt,
715-725.
J. M.,
BIOCHEMISTRY
189,
95-98
(1990)
A Microtiter Plate-Based Assay for Phosphoenolpyruvate Carboxylase William Cockburn,* Garry C. Whitelam,* Stephen P. Slocombe,* and Raymond A. McKee? Botany Department, School of Biological Sciences, University of Leicester, Leicester, LEl 7RH, United Kingdom; and A.F.R.C. Food Research Institute, Colney Lane, Norwich, NR4 7UA, United Kingdom
Received
October
20,1989
A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography. 0 1990 Academic Press, Inc.
achieved by two distinct methods. Nimmo (5) describes a system in which orthophosphate released from PEP in the carboxylation reaction is visualized by precipitation as calcium phosphate. The second and most commonly utilized method relies upon the reaction between oxaloacetate and Fast Violet B, which results in the generation of a colored precipitate (6). The present work describes a sensitive, quantitative method for the assay of PEPC which is based on the Fast Violet reaction and which, because it is carried out in microtiter plates, is particularly suited to the analysis of large numbers of samples. MATERIALS
Phosphoenolpyruvate carboxylase (PEPC)l (EC 4.1.1.31) is usually assayed by coupling the reduction of oxaloacetate produced in the PEPC reaction, by means of malic dehydrogenase (EC 1.1.1.37), to the oxidation of NADH, which is monitored spectrophotometrically (1,2). An improvement in this venerable method, in which pyruvate resulting from the decomposition of oxaloacetate is reduced to lactate by lactate dehydrogenase and therefore contributes to the oxidation of NADH, has recently been suggested (3). Although precise and sensitive this assay is by no means ideal for the monitoring of column eluates or other situations in which large numbers of determinations must be made. PEPC activity has also been measured as the incorporation of radiocarbon from [14C]bicarbonate into oxaloacetate (4). Although very sensitive, and adaptable to use with relatively large numbers of samples, the method suffers from the expense of radioisotopes and the inconvenience and potential hazards of working with volatile radioactive substances. Qualitative localization of PEPC on gels has been ’ Abbreviations used: PEP, phosphoenolpyruvate;
0003-‘2697/QO
Copyright All rights
PEPC, phosphoenolpyruvate TCA, tricarboxylic acid.
$3.00
0 1990 by Academic of reproduction
Press, Inc.
in any form
reserved.
carboxylase;
AND
METHODS
Chemicals and enzymes. Phosphoenolpyruvate, phosphoenolpyruvate carboxylase, malic dehydrogenase, Fast Violet B salt, sodium diethyldithiocarbamate, polyvinylpolypyrrolidone, and Tris buffer were obtained from Sigma Chemical Co. Other chemicals were obtained from BDH Chemicals Ltd., Poole, England. Preparation of crude enzyme extract. Samples of leaf lamina weighing 1.0 g from 12-day-old maize plants were ground to a fine powder in liquid nitrogen. The frozen powder was extracted with 5.0 ml of a solution containing 50 mM Tris/HCl, 30 m&f sodium isoascorbate, 2.0 mM &sodium EDTA, and 1% polyvinylpolypyrrolidone, pH 7.2. The homogenate was centrifuged for 2 min at 12,000 rpm in a bench microfuge and the supernatant solution decanted and stored on ice until required. Aliquots of crude Size exclusion chromatography. enzyme solution (200 ~1) were separated on an LKB GlasPac G3000SW column, using 50 mM Tris/HCl, pH 7.2, containing 2.0 mM dithiothreitol as eluent at a flow rate of 0.4 ml/min. The column eluate was monitored at 280 nm. 95
96
COCKBURN
ET
Spectrophotometric assay. A solution containing 50 Tris/HCl, 10 mM KHCO,, 5.0 mM MgCl,, 2.0 mM PEP, 10 units malic dehydrogenase, 0.2 mM NADH, pH 7.8, was monitored at 340 nm. The reaction was started by the addition of enzyme to bring the final volume to 1.0 ml. Some assays were started by the addition of PEP in order to assess background rates of NADH oxidation. Microtiter plate assay. The final concentrations of reagents in the microtiter plate wells was 100 mM Tris, 40 mM NaHCO,, 10 mM MgCl,, pH 7.2 (added as 25 ~1 of a single stock solution), and 6.0 mM PEP, which was added separately (as 25 ~1 of 24 mM stock solution) to permit the use of minus-substrate controls. The reaction was started by the addition of the enzyme solution, water having been added to ensure the final volume in the well would be 100 ~1. After 10 min incubation with gentle shaking at room temperature (2O“C) 25 ~1 of a 3.0% (wt/vol) solution of Fast Violet B was added to give a final concentration of 0.6%. Following a further lomin incubation the reaction was stopped by the addition of 25 ~1 of 80% lactic acid (vol/vol) to give a final concentration of 13%. After 5.0 min the plate was read at 490 nm using an empty well as blank and the data were expressed as the difference between samples with and without PEP substrate. The color was stable for at least an overnight period. Stock solutions were stored frozen. Once thawed the PEP and Fast Violet solutions were stored on ice.
AL. 0.60
mM
RESULTS
AND
0.46
0.36 z Iii e 2 2 0.24 J
0.12
DISCUSSION
0.00 400
Assay PH. A pH of 7.2 was selected for the standard assay conditions. It was found that the use of a pH greater than 7.8 resulted in the deposition of precipitates in both plus and minus PEP wells which did not redissolve following acidification and which interfered with subsequent optical quantification. As Vidal et al. (6) indicated, mercaptoethanol, isoascorbate, and thioglycollate cause increased background values and are best avoided when possible. Order of addition of reagents. Addition of Fast Violet to the assay after a period of incubation rather than including it from the outset of the assay resulted in enhancement of the difference between plus and minus PEP assays. The reason for this is not known although it is possible that the enzyme is protected from some deleterious effect of Fast Violet by prior incubation with its substrate. The enzyme reaction and the generation of colored product continued in the presence of Fast Violet. Addition of the reagents in the order prescribed also resulted in the deposition of a finely divided precipitate which dissolved readily when the assay was terminated by acidification. Wavelength at which to read assay. Figure 1 shows a difference spectrum of plus and minus PEP replicates
I
I
I
500
600
700
Wavelength FIG. PEP
1. Optical absorbance assay solutions.
difference
(nm) spectrum
of plus
and minus
subjected to the standard assay conditions. The wavelength range between approximately 500 and 550 would be optimal in terms of both magnitude of extinction coefficient and insensitivity to minor wavelength variations. Obviously it is not essential to work within the optimum range and in fact the plate reader, which performed perfectly satisfactorily in the present work, was fitted with a 490-nm filter. Reproducibility of the assay. Analysis of six replicate samples using the standard assay conditions gave a mean value of 0.194 with a sample standard variation of 0.0098. The reproducibility of the assay is therefore satisfactory for most purposes and is certainly more than adequate for monitoring chromatographic eluates. Linearity of response. The linearity of the assay with time, using commercially obtained enzyme, is shown in Fig. 2. Differences between plus and minus PEP wells continued to increase throughout the experiment and
PHOSPHOENOLPYRUVATE
CARBOXYLASE
97
ASSAY
0.06 0.05 s 0.04 al z u 0.03 -
a
0.02 0.01
I
0
I 10
5
I 15 Time
I 20
I 25
I 30
[mid
FIG. 2. Relationship between incubation time and magnitude of difference between optical density reading at 490 nm (AA,& of plus and minus PEP replicate assays. Y = -2.45 X 10m3 (22.26 X 1Om3) + 2.03 X 10e3X (11.24 X 10m4). Determination coefficient, 99.8%.
l1 .t i
I
I 2.4
Elution
although a total incubation time of 20 min was chosen for the standard assay it is clear that incubation periods up to and beyond 30 min are practicable at room temperature. Assays were incubated at room temperature partly for convenience and partly because incubation at higher temperatures increased minus-substrate background levels. Sensitivity of the assay and response to amount of enzyme. The assay was performed with dilutions of com-
0.03 ii P -F
a
0.02
J 0
0.4 PEP carboxylase
0.8
1.2 activity
1.6 units
FIG. 3. Relationship between amounts of enzyme activity present in assay and the difference between optical density readings at 490 nm (AA,& of plus and minus PEP replicate assays. (One unit of enzyme activity catalyzes the synthesis of 1 pmol of oxaloacetate per minute at 20°C.) Y = 2.37 X 10e3 (fl.O1 X lo-‘) + 2.09 X 10m3X (k1.09 X lOma). Determination coefficient, 99.5%.
I volume
I 3.2
I .O (ml)
FIG. 4. Estimation of PEP carboxylase activity by the Fast Violet assay (difference between optical density at 490 nm of plus and minus PEP replicate assays (AA& (0) and optical density at 280 nm (A,) (-) of column eluate fractions from size-exclusion chromatography of a crude extract of leaves of &a nays.
mercial PEPC and it was found that with the incubation times specified it was possible to detect 0.07 units of the enzyme and also that there was a linear response to the amount of enzyme assayed (Fig. 3). The sensitivity observed is similar to that of the conventional spectrophotometric assay. No doubt the sensitivity of both methods could be increased were this a prime consideration. The sensitivity of the standard Fast Violet microtiter plate assay readily permits the measurement of PEPC activity in the column eluate resulting from the size-exclusion chromatography of an unconcentrated crude extract of maize leaves (Fig. 4). Partial separation of PEPC from ribulosebisphosphate carboxylase/oxygenase has been achieved and the enzyme activity coincides with one of the two, partially resolved, protein peaks. The assay is therefore fully satisfactory for the purpose for which it is likely to be most useful-that is the monitoring of chromatographic fractions during purification procedures. PEPC is a component of a number of metabolic pathways including C, and CAM photosynthesis, stomata1 mechanism, anaplerosis in the TCA cycle, and pH regulation. These varied roles impose a range of constraints and requirements on the regulatory characteristics of PEPC, and isoforms of this enzyme, which may well be pathway-specific, are increasingly being discovered
98
COCKBURN
(5,7). Detailed study of these isoforms to determine their kinetic characteristics will inevitably involve chromatographic separation. The microtiter plate assay described here is particularly useful in the monitoring of chromatographic fractions and, while offering equivalent sensitivity, is more convenient and economical than the conventional spectrophotometric assay.
ET
AL.
REFERENCES 1. Bandurski, R. S., and Greiner, 781-786. 2. Walker, D. A. (1957) Biochem. 3. Meyer,
R. C., Rustin,
(1953)
J. 67,
73-79.
P., and Wedding,
J. Biol.
R. T. (1988)
Chem.
Plant
204,
Physiol.
86,325-328. 4. Jones, R., Wilkins, colm,
A. D. B. (1978)
M. B., Coggins, Biochem. J.
5. Nimmo, G. A., Nimmo, J. 239,213-220.
ACKNOWLEDGMENT
C. M.
6. Vidal,
J., Cavalie,
J. R., Fewson,
H. G., and Wilkins,
G., and Vidal,
C. A., and Mal-
175,391-406.
P. (1976)
M. B. (1986) Plant
Sci. Lett.
Biochem. 7,265-
270. The authors are very tion of diagrams.
grateful
to Mrs.
Sue Ogden
for the prepara-
7. Cushman, and Bohnert,
J. C., Meyer, G., Michalowski, H. J. (1989) Plant Cell 1,
C. B., Schmitt,
715-725.
J. M.,