298
GENERAL ANALYTICAL METHODS
[35]
magnitude of change in competitive inhibition occurring after conversion to PGB (Fig. 3). Since PGE's are poor inhibitors of this antibody system, the sample is assayed at a dilution at which PGE's do not cross react. To determine if PGE1 and/or PGE~ are present, the sample can be treated with NaBH4 which quantitatively converts PGE2 to PGF2~ and PGF2~ (approximately at a 50:50 ratio) or PGE1 to PGFI~ and PGF,~. Then the sample can be analyzed with anti-PGF2~ and anti-PGFl~ before and after NaBH4 treatment to establish if PGE1 and/or PGE2 are present. The presence of PGF~a and PGF~a does not present a problem since they crossreact poorly with the respective antisera. If the concentration of PGE's derived from the assay of a NaBH4treated sample is known, this amount is subtracted from the concentration of PGB obtained after alkaline treatment with anti-PGE1 system. The remaining value of PGB represents that amount present in the sample plus PGB converted from PGA. With this particular antiserum it is necessary to separate the individual PGA's and PGB's by reverse phase chromatography27 before assaying in order to accurately quantitate these compounds. The availability of a specific antiserum to PGA would eliminate the need for such a separation. The successful production of highly specific antibodies to PGA1 has been reported. ~s The number of laboratories employing immunologic techniques to determine PG concentrations in biologic systems has been increasing rapidly. The radioimmunoassay has allowed investigators who do not have access to a gas chromatograph mass spectrometer to measure PG's by a relatively simple technique. :' M. I~Iamberg and B. Samuelsson, J. Biol. Chem. 241, 257 (1966). ~ W. A. Stylos and B. Rivetz, Prostaglandins 2, 103 (1972).
[36] E n z y m a t i c D e t e r m i n a t i o n of Microquantities of Acetate I
By MARVIN SCHULMAN and
~-IARLAND G. WOOD
A variety of methods for determination of acetate in biologic fluids have been previously described/-4 but none of these provides a rapid, specific means of determining microquantities of acetate. The method ~ 1 This work was supported in part by USPHS N I H Grant 11839. ~I. A. Rose, M. Grunberg-Manogo, S. R. Carey, and S. Ochoa, J. Biol. Chem. 211, 737 (1954). 3 It. Wierzlicka, A. B. Legacki, and J. Pawekiewicz, Acta Polon. 14 (1967). 4 H. U. Bergmeyer and It. Moellering, Biochem. Z. 344, 167 (1966). M. Schulman and H. G. Wood, Anal. Biochem. 39, 505 (1971).
[361
ENZYMATIC DETERMINATION OF ACETATE
299
to be described is rapid, quite specific, and about twenty times more sensitive than those previously described. Principle. The spectrophotometric determination of acetate involves the conversion of acetate to citrate via the following reactions. CoA-transferase
Succinyl-CoA + acetate , acetyl-CoA + succinate malate dehydrogenase Malate -t- NAD + , oxalacetate + N A D H + H + Oxalacetate + acetyl-CoA
citrate synthase
, citrate + CoA
Succinyl-CoA + acetate + malate -t- NAD+ ~_ citrate -t- succinate + CoA + N A D H + H +
(1) (2) (3)
(4)
Acetate is converted to acetyl-CoA by CoA-transferase from Propionibacterium shermanii [reaction (1)]. The enzyme has an apparent K~ of 7 X 10-a M for acetate2 When coupled with malate dehydrogenase [reaction (2) ] and citrate synthase [reaction (3) ], the conversion of acetate to acetyl-CoA and then to citrate is virtually complete. The amount of acetate is equal to the NADH formed and is measured spectrophotometrically at 340 nm.
Reagents Tris-C1 (Trizma base) (Sigma) 1.0 M pH 8.0 Sodium DL-malate 0.3 M fl-DPN (NAD) (Sigma) 0.02 M D P N H (NADH) 0.005 M Malate dehydrogenase (5 mg of protein per milliliter, 1100 IU/mg of protein, Boehringer) Citrate synthase (2 mg of protein per milliliter, 70 IU/mg of protein, Boehringer) Succinyl-CoA prepared from recrystallized succinic anhydride (J. L. Baker) and CoA (P. L. Biochemicals) by the method of Simon and Shemin r as modified by Swick and Wood2 An approximate determination of succinyl-CoA is made by the hydroxamate method of Lipman and Tuttle 9 at the time of preparation and a final quantitative determination by spectrophotometric assay with CoA-transferase as described by Schulman and Wood. 1° ° S. H. G. Allen, R. W. Kellermeyer, R. L. Stjernholm, and H. G. Wood, J. Bacteriol, 87, 171 (1964). 7 E. J. Simon and D. Shemin, J. Amer. Chem. Soc. 75, 2520 (1953). 8 R. W. Swick and H. G. Wood, Proc. Nat. Acad. Sci. U.S. 42, 28 (1960). F. L i p m a n n and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945). 1oM. Schulman and H. G. Wood, [281.
300
GENERAL ANALYTICAL ~IETI-IODS
[36]
Procedure The determination is conducted in 0.5 ml cuvettes with a 10-nnn light path and 2 mm width at 25 °. Reactants (1) 0.1 ml of Mixture 1 which contains in micromoles per milliliter: Tris-C1 p H 8.0, 25; sodium malate, 7.5; NAD, 20; and N A D H , 0.25. (2) 0.01 ml of Mixture 2 which contains in IU per milliliter: malate dehydrogenase 220 and citrate synthase 35 in 0.1 M phosphate buffer, pH 6.8. (3) 0.01 ml of ~0.014 M succinyl-CoA (4) Acetate to be determined (4-55 nmoles) (5) Sufficient water to bring the volume to 0.24 ml (6) 0.01 ml containing 0.7 unit of CoA-transferase in 0.1 M phosphate buffer, pH 6.8 containing 0.1 m M E D T A . All but the CoA-transferase is added and the initial absorbance is recorded. A blank cuvette containing all the reactants except the acetate sample is prepared for each assay. The initial optical density (OD) of the cuvettes is approximately 0.6 as a result of the N A D H in the mixtures2 ~ The spectrophotometer is adjusted so that the recorder output of the initial absorbance of the cuvettes reads near zero so the full scale of the recorder is available for measurement of absorbance changes. The CoA-transferase is added and the changes in OD recorded with time. The time course of the reaction is shown in Fig. 1. The time required for complete reaction is usually 15-20 minutes. Calculations. The amount of acetate in the sample is calculated as follows using the data of Fig. 1 : ~EB = EB EB0 = 0.255-0.065 = 0.190 AER -- ER -- Ea0 = 0.920-0.040 = 0.880 hEacetate A E R - - AEB = 0.880-0.190 = 0.690 0.690 ~moles acetate = AEacctate/E = - = 0.0277 24.9 -
-
=
~D. J. Pearson [Biochem. J. 95, 23c (1965)] pointed out that this type of determination is subject to substantial error because of differences in the concentration of NADFI at the beginning and end of the assay which result from a change in the relative concentrations of oxalacetate and malate at the beginning and end of the reaction. This error has been reduced by adjusting the concentrations of malate and NAD to appropriate levels and by adding NADH to the initial reaction mixture2
[36l
301
ENZYMATIC DETERMINATION OF ACETATE of CoA Tronsferose
1.0
ER
!
0.9
Q
08 0.7
0 ~ 0.6 I..>. 0.5
Z 0.4 bJ Q
EB
!
.~ 0.3 ¢j ~ 0
0.2
O.I
0
I
2
3
4
5
6
7
8
9 I0
II 12 13 14 15 16 17 18
MIN. Fla. l, Time course of the spectrophotometric determination of acetate. ( A ) Assay cuvette and ( O ) b l a n k cuvette which does not contain acetate. EBo and ERo denote the absorbance of the b l a n k and assay mixture before initiation of reaction by addition of CoA-transferase. E~ and Ea denote absorbances after completion of the reaction. T h i r t y nanomoles of sodium acetate were determined with 0.46 I U CoAtransferase as described in the text. Reproduced from Schulman and Wood," with permission of the publisher.
E is the absorbance of 1 ~mole NADH at 340 nm in 0.25 ml and a 1-cm light path. Accuracy and Sensitivity. The accuracy and sensitivity of the spectrophotometric determination has been determined with standard acetate solutions by Schulman and Wcod. 5 The method is applicable over a wide range of acetate concentrations (5-55 nmoles) and the accuracy generally exceeds 90%. Specificity. The method is quite specific for acetate. Formate, propionate, lactate, pyruvate, butyrate, a-ketobutyrate, succinate, glucose, and methyltetrahydrofolate are without effect. Acetate has been assayed in a complex bacterial growth medium containing yeast extract, tryptone, and tomato juice. 5
GENERAL ANALYTICAL METHODS
[35]
magnitude of change in competitive inhibition occurring after conversion to PGB (Fig. 3). Since PGE's are poor inhibitors of this antibody system, the sample is assayed at a dilution at which PGE's do not cross react. To determine if PGE1 and/or PGE~ are present, the sample can be treated with NaBH4 which quantitatively converts PGE2 to PGF2~ and PGF2~ (approximately at a 50:50 ratio) or PGE1 to PGFI~ and PGF,~. Then the sample can be analyzed with anti-PGF2~ and anti-PGFl~ before and after NaBH4 treatment to establish if PGE1 and/or PGE2 are present. The presence of PGF~a and PGF~a does not present a problem since they crossreact poorly with the respective antisera. If the concentration of PGE's derived from the assay of a NaBH4treated sample is known, this amount is subtracted from the concentration of PGB obtained after alkaline treatment with anti-PGE1 system. The remaining value of PGB represents that amount present in the sample plus PGB converted from PGA. With this particular antiserum it is necessary to separate the individual PGA's and PGB's by reverse phase chromatography27 before assaying in order to accurately quantitate these compounds. The availability of a specific antiserum to PGA would eliminate the need for such a separation. The successful production of highly specific antibodies to PGA1 has been reported. ~s The number of laboratories employing immunologic techniques to determine PG concentrations in biologic systems has been increasing rapidly. The radioimmunoassay has allowed investigators who do not have access to a gas chromatograph mass spectrometer to measure PG's by a relatively simple technique. :' M. I~Iamberg and B. Samuelsson, J. Biol. Chem. 241, 257 (1966). ~ W. A. Stylos and B. Rivetz, Prostaglandins 2, 103 (1972).
[36] E n z y m a t i c D e t e r m i n a t i o n of Microquantities of Acetate I
By MARVIN SCHULMAN and
~-IARLAND G. WOOD
A variety of methods for determination of acetate in biologic fluids have been previously described/-4 but none of these provides a rapid, specific means of determining microquantities of acetate. The method ~ 1 This work was supported in part by USPHS N I H Grant 11839. ~I. A. Rose, M. Grunberg-Manogo, S. R. Carey, and S. Ochoa, J. Biol. Chem. 211, 737 (1954). 3 It. Wierzlicka, A. B. Legacki, and J. Pawekiewicz, Acta Polon. 14 (1967). 4 H. U. Bergmeyer and It. Moellering, Biochem. Z. 344, 167 (1966). M. Schulman and H. G. Wood, Anal. Biochem. 39, 505 (1971).
[361
ENZYMATIC DETERMINATION OF ACETATE
299
to be described is rapid, quite specific, and about twenty times more sensitive than those previously described. Principle. The spectrophotometric determination of acetate involves the conversion of acetate to citrate via the following reactions. CoA-transferase
Succinyl-CoA + acetate , acetyl-CoA + succinate malate dehydrogenase Malate -t- NAD + , oxalacetate + N A D H + H + Oxalacetate + acetyl-CoA
citrate synthase
, citrate + CoA
Succinyl-CoA + acetate + malate -t- NAD+ ~_ citrate -t- succinate + CoA + N A D H + H +
(1) (2) (3)
(4)
Acetate is converted to acetyl-CoA by CoA-transferase from Propionibacterium shermanii [reaction (1)]. The enzyme has an apparent K~ of 7 X 10-a M for acetate2 When coupled with malate dehydrogenase [reaction (2) ] and citrate synthase [reaction (3) ], the conversion of acetate to acetyl-CoA and then to citrate is virtually complete. The amount of acetate is equal to the NADH formed and is measured spectrophotometrically at 340 nm.
Reagents Tris-C1 (Trizma base) (Sigma) 1.0 M pH 8.0 Sodium DL-malate 0.3 M fl-DPN (NAD) (Sigma) 0.02 M D P N H (NADH) 0.005 M Malate dehydrogenase (5 mg of protein per milliliter, 1100 IU/mg of protein, Boehringer) Citrate synthase (2 mg of protein per milliliter, 70 IU/mg of protein, Boehringer) Succinyl-CoA prepared from recrystallized succinic anhydride (J. L. Baker) and CoA (P. L. Biochemicals) by the method of Simon and Shemin r as modified by Swick and Wood2 An approximate determination of succinyl-CoA is made by the hydroxamate method of Lipman and Tuttle 9 at the time of preparation and a final quantitative determination by spectrophotometric assay with CoA-transferase as described by Schulman and Wood. 1° ° S. H. G. Allen, R. W. Kellermeyer, R. L. Stjernholm, and H. G. Wood, J. Bacteriol, 87, 171 (1964). 7 E. J. Simon and D. Shemin, J. Amer. Chem. Soc. 75, 2520 (1953). 8 R. W. Swick and H. G. Wood, Proc. Nat. Acad. Sci. U.S. 42, 28 (1960). F. L i p m a n n and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945). 1oM. Schulman and H. G. Wood, [281.
300
GENERAL ANALYTICAL ~IETI-IODS
[36]
Procedure The determination is conducted in 0.5 ml cuvettes with a 10-nnn light path and 2 mm width at 25 °. Reactants (1) 0.1 ml of Mixture 1 which contains in micromoles per milliliter: Tris-C1 p H 8.0, 25; sodium malate, 7.5; NAD, 20; and N A D H , 0.25. (2) 0.01 ml of Mixture 2 which contains in IU per milliliter: malate dehydrogenase 220 and citrate synthase 35 in 0.1 M phosphate buffer, pH 6.8. (3) 0.01 ml of ~0.014 M succinyl-CoA (4) Acetate to be determined (4-55 nmoles) (5) Sufficient water to bring the volume to 0.24 ml (6) 0.01 ml containing 0.7 unit of CoA-transferase in 0.1 M phosphate buffer, pH 6.8 containing 0.1 m M E D T A . All but the CoA-transferase is added and the initial absorbance is recorded. A blank cuvette containing all the reactants except the acetate sample is prepared for each assay. The initial optical density (OD) of the cuvettes is approximately 0.6 as a result of the N A D H in the mixtures2 ~ The spectrophotometer is adjusted so that the recorder output of the initial absorbance of the cuvettes reads near zero so the full scale of the recorder is available for measurement of absorbance changes. The CoA-transferase is added and the changes in OD recorded with time. The time course of the reaction is shown in Fig. 1. The time required for complete reaction is usually 15-20 minutes. Calculations. The amount of acetate in the sample is calculated as follows using the data of Fig. 1 : ~EB = EB EB0 = 0.255-0.065 = 0.190 AER -- ER -- Ea0 = 0.920-0.040 = 0.880 hEacetate A E R - - AEB = 0.880-0.190 = 0.690 0.690 ~moles acetate = AEacctate/E = - = 0.0277 24.9 -
-
=
~D. J. Pearson [Biochem. J. 95, 23c (1965)] pointed out that this type of determination is subject to substantial error because of differences in the concentration of NADFI at the beginning and end of the assay which result from a change in the relative concentrations of oxalacetate and malate at the beginning and end of the reaction. This error has been reduced by adjusting the concentrations of malate and NAD to appropriate levels and by adding NADH to the initial reaction mixture2
[36l
301
ENZYMATIC DETERMINATION OF ACETATE of CoA Tronsferose
1.0
ER
!
0.9
Q
08 0.7
0 ~ 0.6 I..>. 0.5
Z 0.4 bJ Q
EB
!
.~ 0.3 ¢j ~ 0
0.2
O.I
0
I
2
3
4
5
6
7
8
9 I0
II 12 13 14 15 16 17 18
MIN. Fla. l, Time course of the spectrophotometric determination of acetate. ( A ) Assay cuvette and ( O ) b l a n k cuvette which does not contain acetate. EBo and ERo denote the absorbance of the b l a n k and assay mixture before initiation of reaction by addition of CoA-transferase. E~ and Ea denote absorbances after completion of the reaction. T h i r t y nanomoles of sodium acetate were determined with 0.46 I U CoAtransferase as described in the text. Reproduced from Schulman and Wood," with permission of the publisher.
E is the absorbance of 1 ~mole NADH at 340 nm in 0.25 ml and a 1-cm light path. Accuracy and Sensitivity. The accuracy and sensitivity of the spectrophotometric determination has been determined with standard acetate solutions by Schulman and Wcod. 5 The method is applicable over a wide range of acetate concentrations (5-55 nmoles) and the accuracy generally exceeds 90%. Specificity. The method is quite specific for acetate. Formate, propionate, lactate, pyruvate, butyrate, a-ketobutyrate, succinate, glucose, and methyltetrahydrofolate are without effect. Acetate has been assayed in a complex bacterial growth medium containing yeast extract, tryptone, and tomato juice. 5
