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An enzymatic assay for acetate in spent bacterial culture supernatants
ANALYTICALBIOCHEMISTRY
13@402-405(1983)
An Enzymatic Assay for Acetate in Spent Bacterial Culture Supernatants PETER M. CLARKE AND MARK A. PAYTON Department
of Biochemistry,
Imperial
College
of Science
and Technology,
London
SW7 2 AZ,
United
Kingdom
Received September 9, 1982 A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supematants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et ai. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 pM. The assay is cheaper than commercially available assaysand is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assayto the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs. KEY WORDS: bacterial fermentation; acetate assay; acetate kinase.
Many laboratories which are routinely involved in assaying acetate levels in spent bacterial culture supematants or in the brewing, food, or dairy industries, frequently do so using gas chromatographic techniques (2). There are, however, situations when insufficient assays are performed to justify the expense and time for purchase and setting up of gas chromatographic equipment. Under such circumstances, one obvious alternative is the use of an enzymatic assay for the determination of acetate. We have found, however, that previously published (3) and commercially available enzymatic assays for acetate are complicated and expensive. We therefore sought to develop an alternative acetate assay for occasional use based on a previously reported assay for acetate kinase (1). The assay involves the conversion of acetate to acetyl-phosphate using acetate kinase and ATP and measuring the ADP produced by a pyruvate kinase/lactate dehydrogenase couple, facilitating the direct spectrophotometric assay at 365 or 340 nm of NADH disappearance (Fig. 1). This assay is cheaper and more convenient than currently available assays and unlike the preceding indicator reaction (3), produces a linear standard plot. We here describe the assay, and in particular its application to the 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
402
measurement of acetate in spent bacterial culture supematants. MATERIALS
AND METHODS
Reagents. All chemicals used were analytical grade obtained from BDH Chemicals Ltd. Poole, United Kingdom, or Fisons, Loughborough, Leicester, United Kingdom. ATP, NADH, phosphoenol pyruvate (PEP),’ and all enzyme preparations were obtained from Sigma Chemical Company Ltd., Poole, United Kingdom. Acetate assay kits, based on the preceding indicator reaction, were obtained from Boehringer Corporation (Lewes, United Kingdom). Growth of bacteria and samplepreparation. Escherichia coli IY 13 (4), JRG106 1,
JRG 1078, JRG 1086 (5), and CSH62 (6) were grown overnight at 37°C in bubble tubes of L Broth (i.e., 10 g Bacto tryptone, 5 g yeast extract, 10 g NaCl/liter distilled water) containing 0.2% glucose. Samples were harvested by centrifugation at 2000 rpm for 10 min in a benchtop centrifuge; the supematant was assayed immediately or stored frozen. For gas ’ Abbreviation used: PEP, phosphoenol pyruvate.
ENZYMATIC
PEP
ACETYL-@
PYRUVATE
ACETATE
ACETATE
NADH
LACTATE DEHYDROGENASE
C
NAD
1
LACTATE FIG. 1. Schematic diagram of the acetate on the acetate kinase reaction.
assay based
chromatographic analysis, the samples were methylated by the procedure used by Drummond and Shama (2). Acetate standards were prepared by dissolving sodium acetate in distilled, deionized water. Apparatus. Spectrophotometric determinations were performed on a Cat-y 2 10 double-beam spectrophotometer. Gas chromato-
ASSAY
403
graphic determinations were performed on a Pye Unicam GCD gas chromatograph equipped with dual-flame ionization detectors. Assay procedure. The assay mixture contained the following in a final volume of 3.02 ml (final concentration in parentheses): potassium phosphate buffer, pH 7.6 (152 mM); MgClz (20 mM); PEP (0.5 mM); NADH (0.5 mM); ATP (5.0 mM); pyruvate kinase/lactate dehydrogenase (4.0 and 5.7 U/ml, respectively); sample (8-160 PM acetate). The reagents were mixed and allowed to equilibrate for 10 min at 37°C. After noting the absorbance at 365 nm (A,), 20 ~1 of acetate kinase were added to give a final concentration to 1.3 U/ml. The reagents were again mixed and incubated at 37°C for 60 min before noting the final absorbance at 365 nm (Al). The change in absorbance (AFI = A2 - A,) could then be calculated and related to concentration by reading from a standard plot.
FIG. 2. Relationship of acetate concentration in nanomoles per cuvette volume (3.02 ml) to AA, the change in absorbance at 365 nm, showing pH-dependent variation in the progress of the reaction. 0, pH 8.0; 0, pH 7.6; A. pH 7.0; 0, pH 6.5. ’
404
CLARKE
0
I SO
I 100
I 150
AND PAYTON
I 200 nMoles
I 250
I 300
I 350
I LOO
I 450
I 500
ACETATE
FIG. 3. Effect of 1.5 mM pyruvate in acetate standards upon the linearity of the standard plot. 0, Normal NADH concentration; 0, double NADH concentration. RESULTS
AND
DISCUSSION
Appropriate dilutions of bacterial supernatants are assayed as described under Materials and Methods and the acetate levels can be read directly off a standard curve prepared from identically treated standard acetate solutions. A typical standard plot is shown in Fig. 2. The assay is sufficiently sensitive easily to detect acetate levels as low as 50 PM. Figure 2 also shows the effect of buffer pH on the An optiapparent u365 nm values obtained. mum pH of 7.6 was adopted as it lay in the middle of the pH range that permitted both the reaction to proceed to completion (within the reaction time of 60 min) and a reproducibly linear standard plot. The assay is also very specific; the only reported side specificity of this acetate kinase from E. coli is towards propionate which exhibits only 10% of the activity with acetate (7). However, it should be noted that high levels of pyruvate in culture supernatants may interfere with the assay. The inclusion of a reaction blank lacking
acetate kinase will indicate pyruvate levels and therefore change in absorbance due to this may be taken into account. If pyruvate levels are exceptionally high, however (i.e., those levels resulting in an initial A365,,“, below 0.80) the addition of higher concentrations of NADH may be necessary to maintain NADH in excess in the cuvette. Figure 3 shows the effect of the addition of pyruvate (1.5 mM final concentration) to a set of acetate standards. It can be seen that in TABLE 1 APPARENT LEVELS OF ACETATE IN THE SPENT CULTURE MEDIUM OF E. coli IY 13 DETERMINED BY THREE DIFFERENT METHODS
Sample IY13 IY13 IY13
Assay method This work Gas chromatography Boehringer-Mannheim assay kit
Apparent acetate concentration m4 26 23 26
ENZYMATIC
ACETATE
TABLE 2 APPARENT LEVELS OF ACETATE IN THE SPENT CULTURE MEDIUM OF MUTANT STRAINS OF E. co/i
Strain CSH JRG JRG JRG
62 1061 1078 1086
Relevant genotype
Acetate concentration (mM)
w/t
15.0
ack-
9.0
&A
1.0
pla-
0
this example at higher acetate concentrations (above 250 nmol per cuvette) there is a deviation from the normal linear plot due to depletion of NADH, resulting in insufficient amounts of NADH to allow the coupled acetate-dependent reaction to proceed to completion. This can be compensated, however, by adding more NADH to the assay mixture; in Fig. 3 the effect is of a doubling of NADH concentration. Alternatively, the problem of “pyruvate interference” should be both indicated and overcome by routinely assaying appropriate dilutions of the unknown sample. In order to compare the results of this assay with other available methods, samples prepared from overnight cultures of E. cd IY 13 were assayed by three different methods; the assay described here, gas chromatography (2) and the “preceding indicator reaction” marketed by Boehringer-Mannheim. The results are shown in Table 1 and demonstrate the similarity in measured acetate concentrations from the three methods. Determinations of acetate levels in spent culture supematants of strains harboring mutations in those genes responsible for acetate formation have also been undertaken. The consequences of these mutations should be that acetate kinase (a&)-deficient strains (e.g., JRG 106 1) show reduced acetate levels, while phosphotransacetylase (p&-)-deficient strains and facA strains deficient in both acetate kinase and phosphotransacetylase activities (e.g., JRG1086 and JRG1078, respectively) show little or no acetate production (8). The results in Table 2, obtained by our assay procedure show that this is so.
ASSAY
405
Those problems associated with high levels of pyruvate have not been encountered in our work with E. coli and therefore we have used the assay as described in this paper. However, it is probable that when this assay is applied to certain other organisms (e.g., Bacillus stearothermophilus), it will become necessary to increase the NADH concentration to overcome depletion due to high levels of pyruvate in the samples or to assay a range of sample dilutions. This assay has been routinely used in our laboratory to determine acetate production by many strains of E. coli and other organisms, consistently giving values identical with those obtained by the other two assay procedures mentioned but more rapidly and at lower cost. Although here we have only described the application of the assay to the determination of acetate levels in spent bacterial culture supematants, the assay should be valid for a wide range of biological fluids and in the food, brewing, and dairy industries. ACKNOWLEDGMENTS Special thanks to G. Shama for help and advice in the use of gas chromatographic equipment, and to Professor J. R. Guest for kindly supplying us with bacterial strains. This work was supported by an MRC research studentship (P.M.C.) and by Biogen N.V. (M.A.P.). REFERENCES 1. Bergmeyer, H. U., Cawehn, K., and Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer. H. U., ed.), Vol. 1, pp. 425-426, Verlag ChemieAcademic Press, New York/London. 2. Drummond, I., and Shama, G. (1982) Chromatographia
15, 180-182.
3. Bergmeyer, H. U., and Mollering, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 1520-1537. Verlag Chemie-Academic Press, New York/London. 4. Young. I. G., and Wallace, B. J. (1976) Biochim. Biophys.
Acfa 499, 376-385.
Guest, J. R. (1979) J. Gem Microbial. 155,259-27 I. 6. Miller, J. H. (1972) in Experiments in Molecular Genetics, p. 2 1, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 7. Rose, I. A., Grunberg-Manago, M., Korey, S. R., and Ochoa, S. (1954) J. Biol. Chem. 211, 737-756. 8. Brown, T. D. K., Jones-Mortimer, M. C., and Kornberg, H. L. (1977) J. Gen. Microbial. 102, 3275.
336.
13@402-405(1983)
An Enzymatic Assay for Acetate in Spent Bacterial Culture Supernatants PETER M. CLARKE AND MARK A. PAYTON Department
of Biochemistry,
Imperial
College
of Science
and Technology,
London
SW7 2 AZ,
United
Kingdom
Received September 9, 1982 A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supematants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et ai. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 pM. The assay is cheaper than commercially available assaysand is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assayto the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs. KEY WORDS: bacterial fermentation; acetate assay; acetate kinase.
Many laboratories which are routinely involved in assaying acetate levels in spent bacterial culture supematants or in the brewing, food, or dairy industries, frequently do so using gas chromatographic techniques (2). There are, however, situations when insufficient assays are performed to justify the expense and time for purchase and setting up of gas chromatographic equipment. Under such circumstances, one obvious alternative is the use of an enzymatic assay for the determination of acetate. We have found, however, that previously published (3) and commercially available enzymatic assays for acetate are complicated and expensive. We therefore sought to develop an alternative acetate assay for occasional use based on a previously reported assay for acetate kinase (1). The assay involves the conversion of acetate to acetyl-phosphate using acetate kinase and ATP and measuring the ADP produced by a pyruvate kinase/lactate dehydrogenase couple, facilitating the direct spectrophotometric assay at 365 or 340 nm of NADH disappearance (Fig. 1). This assay is cheaper and more convenient than currently available assays and unlike the preceding indicator reaction (3), produces a linear standard plot. We here describe the assay, and in particular its application to the 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
402
measurement of acetate in spent bacterial culture supematants. MATERIALS
AND METHODS
Reagents. All chemicals used were analytical grade obtained from BDH Chemicals Ltd. Poole, United Kingdom, or Fisons, Loughborough, Leicester, United Kingdom. ATP, NADH, phosphoenol pyruvate (PEP),’ and all enzyme preparations were obtained from Sigma Chemical Company Ltd., Poole, United Kingdom. Acetate assay kits, based on the preceding indicator reaction, were obtained from Boehringer Corporation (Lewes, United Kingdom). Growth of bacteria and samplepreparation. Escherichia coli IY 13 (4), JRG106 1,
JRG 1078, JRG 1086 (5), and CSH62 (6) were grown overnight at 37°C in bubble tubes of L Broth (i.e., 10 g Bacto tryptone, 5 g yeast extract, 10 g NaCl/liter distilled water) containing 0.2% glucose. Samples were harvested by centrifugation at 2000 rpm for 10 min in a benchtop centrifuge; the supematant was assayed immediately or stored frozen. For gas ’ Abbreviation used: PEP, phosphoenol pyruvate.
ENZYMATIC
PEP
ACETYL-@
PYRUVATE
ACETATE
ACETATE
NADH
LACTATE DEHYDROGENASE
C
NAD
1
LACTATE FIG. 1. Schematic diagram of the acetate on the acetate kinase reaction.
assay based
chromatographic analysis, the samples were methylated by the procedure used by Drummond and Shama (2). Acetate standards were prepared by dissolving sodium acetate in distilled, deionized water. Apparatus. Spectrophotometric determinations were performed on a Cat-y 2 10 double-beam spectrophotometer. Gas chromato-
ASSAY
403
graphic determinations were performed on a Pye Unicam GCD gas chromatograph equipped with dual-flame ionization detectors. Assay procedure. The assay mixture contained the following in a final volume of 3.02 ml (final concentration in parentheses): potassium phosphate buffer, pH 7.6 (152 mM); MgClz (20 mM); PEP (0.5 mM); NADH (0.5 mM); ATP (5.0 mM); pyruvate kinase/lactate dehydrogenase (4.0 and 5.7 U/ml, respectively); sample (8-160 PM acetate). The reagents were mixed and allowed to equilibrate for 10 min at 37°C. After noting the absorbance at 365 nm (A,), 20 ~1 of acetate kinase were added to give a final concentration to 1.3 U/ml. The reagents were again mixed and incubated at 37°C for 60 min before noting the final absorbance at 365 nm (Al). The change in absorbance (AFI = A2 - A,) could then be calculated and related to concentration by reading from a standard plot.
FIG. 2. Relationship of acetate concentration in nanomoles per cuvette volume (3.02 ml) to AA, the change in absorbance at 365 nm, showing pH-dependent variation in the progress of the reaction. 0, pH 8.0; 0, pH 7.6; A. pH 7.0; 0, pH 6.5. ’
404
CLARKE
0
I SO
I 100
I 150
AND PAYTON
I 200 nMoles
I 250
I 300
I 350
I LOO
I 450
I 500
ACETATE
FIG. 3. Effect of 1.5 mM pyruvate in acetate standards upon the linearity of the standard plot. 0, Normal NADH concentration; 0, double NADH concentration. RESULTS
AND
DISCUSSION
Appropriate dilutions of bacterial supernatants are assayed as described under Materials and Methods and the acetate levels can be read directly off a standard curve prepared from identically treated standard acetate solutions. A typical standard plot is shown in Fig. 2. The assay is sufficiently sensitive easily to detect acetate levels as low as 50 PM. Figure 2 also shows the effect of buffer pH on the An optiapparent u365 nm values obtained. mum pH of 7.6 was adopted as it lay in the middle of the pH range that permitted both the reaction to proceed to completion (within the reaction time of 60 min) and a reproducibly linear standard plot. The assay is also very specific; the only reported side specificity of this acetate kinase from E. coli is towards propionate which exhibits only 10% of the activity with acetate (7). However, it should be noted that high levels of pyruvate in culture supernatants may interfere with the assay. The inclusion of a reaction blank lacking
acetate kinase will indicate pyruvate levels and therefore change in absorbance due to this may be taken into account. If pyruvate levels are exceptionally high, however (i.e., those levels resulting in an initial A365,,“, below 0.80) the addition of higher concentrations of NADH may be necessary to maintain NADH in excess in the cuvette. Figure 3 shows the effect of the addition of pyruvate (1.5 mM final concentration) to a set of acetate standards. It can be seen that in TABLE 1 APPARENT LEVELS OF ACETATE IN THE SPENT CULTURE MEDIUM OF E. coli IY 13 DETERMINED BY THREE DIFFERENT METHODS
Sample IY13 IY13 IY13
Assay method This work Gas chromatography Boehringer-Mannheim assay kit
Apparent acetate concentration m4 26 23 26
ENZYMATIC
ACETATE
TABLE 2 APPARENT LEVELS OF ACETATE IN THE SPENT CULTURE MEDIUM OF MUTANT STRAINS OF E. co/i
Strain CSH JRG JRG JRG
62 1061 1078 1086
Relevant genotype
Acetate concentration (mM)
w/t
15.0
ack-
9.0
&A
1.0
pla-
0
this example at higher acetate concentrations (above 250 nmol per cuvette) there is a deviation from the normal linear plot due to depletion of NADH, resulting in insufficient amounts of NADH to allow the coupled acetate-dependent reaction to proceed to completion. This can be compensated, however, by adding more NADH to the assay mixture; in Fig. 3 the effect is of a doubling of NADH concentration. Alternatively, the problem of “pyruvate interference” should be both indicated and overcome by routinely assaying appropriate dilutions of the unknown sample. In order to compare the results of this assay with other available methods, samples prepared from overnight cultures of E. cd IY 13 were assayed by three different methods; the assay described here, gas chromatography (2) and the “preceding indicator reaction” marketed by Boehringer-Mannheim. The results are shown in Table 1 and demonstrate the similarity in measured acetate concentrations from the three methods. Determinations of acetate levels in spent culture supematants of strains harboring mutations in those genes responsible for acetate formation have also been undertaken. The consequences of these mutations should be that acetate kinase (a&)-deficient strains (e.g., JRG 106 1) show reduced acetate levels, while phosphotransacetylase (p&-)-deficient strains and facA strains deficient in both acetate kinase and phosphotransacetylase activities (e.g., JRG1086 and JRG1078, respectively) show little or no acetate production (8). The results in Table 2, obtained by our assay procedure show that this is so.
ASSAY
405
Those problems associated with high levels of pyruvate have not been encountered in our work with E. coli and therefore we have used the assay as described in this paper. However, it is probable that when this assay is applied to certain other organisms (e.g., Bacillus stearothermophilus), it will become necessary to increase the NADH concentration to overcome depletion due to high levels of pyruvate in the samples or to assay a range of sample dilutions. This assay has been routinely used in our laboratory to determine acetate production by many strains of E. coli and other organisms, consistently giving values identical with those obtained by the other two assay procedures mentioned but more rapidly and at lower cost. Although here we have only described the application of the assay to the determination of acetate levels in spent bacterial culture supematants, the assay should be valid for a wide range of biological fluids and in the food, brewing, and dairy industries. ACKNOWLEDGMENTS Special thanks to G. Shama for help and advice in the use of gas chromatographic equipment, and to Professor J. R. Guest for kindly supplying us with bacterial strains. This work was supported by an MRC research studentship (P.M.C.) and by Biogen N.V. (M.A.P.). REFERENCES 1. Bergmeyer, H. U., Cawehn, K., and Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer. H. U., ed.), Vol. 1, pp. 425-426, Verlag ChemieAcademic Press, New York/London. 2. Drummond, I., and Shama, G. (1982) Chromatographia
15, 180-182.
3. Bergmeyer, H. U., and Mollering, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 1520-1537. Verlag Chemie-Academic Press, New York/London. 4. Young. I. G., and Wallace, B. J. (1976) Biochim. Biophys.
Acfa 499, 376-385.
Guest, J. R. (1979) J. Gem Microbial. 155,259-27 I. 6. Miller, J. H. (1972) in Experiments in Molecular Genetics, p. 2 1, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 7. Rose, I. A., Grunberg-Manago, M., Korey, S. R., and Ochoa, S. (1954) J. Biol. Chem. 211, 737-756. 8. Brown, T. D. K., Jones-Mortimer, M. C., and Kornberg, H. L. (1977) J. Gen. Microbial. 102, 3275.
336.