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Determination of pyruvate kinase activity by recording of pH changes
ANALYTICAL
38, 309-312
BIOCHEMISTRY
Determination
of Pyruvate
by Recording C. CARMELI Department
of
(1970)
Biochemistry,
Received
Kinase
Activity
of pH Changes AND
Y. LIFSHITZ
Tel-Aviv
February
University,
Tel-Aviv,
Israel
18, 1970
The activity of PK (pyruvate kinase) is generally followed by enzymic assay of pyruvate. The oxidation of NADH during the conversion of pyruvate to lactate by lactic dehydrogenase is followed spectroscopically (1). An inherent limitation of this method arises from the use of coupling enzymes for the determination of PK activity. We have developed a sensitive method for the direct measurement of PK activity by a continuous recording of the change in proton concentration which accompanies the conversion of PEP (phosphoenolpyruvate) and ADP to ATP. This method was developed after the method for the measurement of ATP synthesis during photophosphorylation (2). Determination of PK activity by recording of PH changes was found by us to be much more sensitive than the measurement of the activity by recording of the titration of the hydroxyl ions released by this process (3, 4). METHODS
Principles
The synthesis of ATP from PEP and ADP by PK is accompanied by an uptake of protons from the medium at a physiological pH range. The reaction proceeds as follows: (PEP-
t-f PEPa-) + (ADP”
t* ADP3-) + nH+ + (ATPa- c-t ATP4-)
+ pyruvatel-
In calculations of the n value we used the pK,’ values for the last dissociable group of the magnesium complexes of ATP and ADP. The pKa’ values, at 25’, 0.2 ionic strength, for magnesium ATP and ADP were 4.97 and 5.13, respectively (5). For PEP, pK,’ 5.82 was determined at the same ionic strenght as was used in the determination of PK activity. Using the pKa’ values given above at 7.5, a value of n = 0.96 was calculated. Since n = AH+ taken up per ATP synthesized, the rate of ATP formation can be calculated from the n value obtained at any given pH. 309
@ 1970 by Academic
Preaa, Inc.
310
CARMELI
AND
LIFSHITZ
Reaction Mixture For spectroscopic assay of PK activity, reaction conditions were similar to those used in the method of Bucher (1). In most of the experiments the reaction mixture contained: KCI, 75 mM; Tris-HCI, pH 7.5, 30 mM; MgCl,, 8 mM; PEP, 0.8 mM; NADH, 0.43 mM; ADP, 0.27 mM; lactate dehydrogenase, 20 pg/ml in a total volume of 3 ml at temperature of 23 T 2”. For determination of ADP 0.6 I.U. of PK was used. The reaction was started with the addition of 140 ~1 ADP solution containing Mg,CI, in a ratio of 1:2. For pH measurements the reaction mixture was the same as above except for the omission of NADH and lactate dehydrogenase and for the buffer concentration which was reduced to 1 mM Tris-HCl. HCI, 0.01 IV, was used for determination of the buffer capacity of the reaction mixture. For measurements by titrator the reaction mixture was the same as for pH measurements except for the following variations: in the determination of ADP and PEP Tris buffer was omitted and 0.01 N H.,SO, was used for titration; in the assay of PK activity the concentrations of PEP and ADP were doubled and 0.02 N H&JO4 was used for titration. All acid solutions were standardized against a concentrated volumetric HCl solution purchased from British Drug Houses, Ltd. Apparatus pH changes were measured in a 5 ml cylindrical glass cuvet. The solution was rapidly stirred by a small magnetic stirrer. A Radiometer KG2026C pH electrode was used for pH measurements. The electrode was connected to a Radiometer model 26 pH meter and the recording was done by a Sargent SRG recorder equipped with an auxiliary zero suppression device. Titration was performed by a Radiometer titrator equipped with a pH meter model 26, a titrator model II, a titrograph, and a KG2026C pH electrode. A proportional band of 0.2 was selected, with a syringe buret motor speed of 20 rpm. Spectroscopic measurements were carried out by a Hitachi PerkinElmer model 124 double-beam recording spectrophotometer. RESULTS
AND
DISCUSSION
A recording of the pH changes during PK activity is given in Figure 1. On the addition of ADP aa increase in pH was recorded. It was assumed that the rate of the pH change depended on the rate of PK activity and that the end of the reaction was indicated by the end of the increase
PH
DETERMINATION
OF
PYRUVATE
311
KINASE
- 60 t]’ HCI
0.01 N
FIQ. 1. Recording of pH changes during PK activity with (A) PK present in the medium and (B) PK omitted from the medium. Experimental conditions as described under “Methods.” The pH meter output was 10 mV per 1 unit pH and the recorder was set on 5 mV giving a full scale recording of 0.5 unit pH.
in pH. With limiting amounts of ADP there was almost complete phosphorylation of ADP to ATP at the end of the reaction. Similar changes in pH (not shown here) were recorded when the reaction was started with the addition of PK or PEP instead of ADP, thus indicating that the changes in pH were due to PK activity. The buffer capacity was determined by titration with HCl. Determination of the buffer capacity of the reaction mixture over the same pH range which changed during the reaction ensured accurate results. The initial rate of PK activity was determined from the linear part of the slope at the beginning of the recording. The amount of ADP was determined from the extent of the pH change (the difference between the pH at the beginning and at the end of the reaction). There was a linear relation between the amount of ADP added and the amount measured by this method between 0.1 and 0.7 pmole ADP (Fig. 2). By reducing the buffer concentration and reducing the impedance of the recorder to 1 mV, as little as 0.01 pmole ADP could be measured accurately. Similar results were obtained when the ADP content of a given solution was determined by measuring either changes in pH or titration of hydroxyl ion by the spectroscopic method (Fig, 2). The method revealed a linear relation between the changes in enzyme concentration and the changes in the activity measured (not shown). By reducing the buffer concentration and by increasing the recorder sensitivity as little as 0.03 I.U. of PK activity could be measured. The rate of the change in pH during PK activity was assumed to reflect
312
CARMELI
AND
ADP
LIFSHITZ
pmoles
addedxl0)
FAG. 2. Determination of ADP by three different methods: (0) recording of pH changes, (A) titration with a titrator. ditions so described under “Methods.”
-(0) spectroscopic, Experimental con-
the actual kinetics of the activity because hydroxyl ions are one of the products of the reaction. The quick addition o.f the substrate, the rapid stirring of the solution, and the immediate recording of changes in pH promised reliable results. Recording of pH was found to be 20-fold more sensitive than direct titration both in the measurement of PK activity and in the determination of ADP. SUMMARY
Pyruvate kinase activity was determined by recording changes in pH and by recording titration of the hydroxyl ions released during the reaction. The same methods were also employed for determination of ADP and of phosphoenolpyruvate. The results obtained in the determination of pyruvate kinase activity by following the pH changes and by titration were compared to those obtained by the spectroscopic method using lactate dehydrogenase and NADH. REFERENCES 1. BUCXER, T, in “Methods in Enzymology” (9. P. Colowick and N. 0. Kaplan, eds.), Vol. 1, p. 435. Academic Press, New York, 1955. 2. NISHIMURA, M., ITO, T., AND CHANCE, B., Biochim. Biophys. Acta 59, 177 (1962). 3. KAYNE, F. J., AND SUELTER, C. H., J. Am. Chem. Sot. 87, 897 (1956). 4. MELCHIOR, J. B., Biochemistry 4, 1518 (1965). 5. SMITH, R. M., AND ALBEETY, R. A., .I. Am. Chem. Sac. 78, 2376 (1956).
38, 309-312
BIOCHEMISTRY
Determination
of Pyruvate
by Recording C. CARMELI Department
of
(1970)
Biochemistry,
Received
Kinase
Activity
of pH Changes AND
Y. LIFSHITZ
Tel-Aviv
February
University,
Tel-Aviv,
Israel
18, 1970
The activity of PK (pyruvate kinase) is generally followed by enzymic assay of pyruvate. The oxidation of NADH during the conversion of pyruvate to lactate by lactic dehydrogenase is followed spectroscopically (1). An inherent limitation of this method arises from the use of coupling enzymes for the determination of PK activity. We have developed a sensitive method for the direct measurement of PK activity by a continuous recording of the change in proton concentration which accompanies the conversion of PEP (phosphoenolpyruvate) and ADP to ATP. This method was developed after the method for the measurement of ATP synthesis during photophosphorylation (2). Determination of PK activity by recording of PH changes was found by us to be much more sensitive than the measurement of the activity by recording of the titration of the hydroxyl ions released by this process (3, 4). METHODS
Principles
The synthesis of ATP from PEP and ADP by PK is accompanied by an uptake of protons from the medium at a physiological pH range. The reaction proceeds as follows: (PEP-
t-f PEPa-) + (ADP”
t* ADP3-) + nH+ + (ATPa- c-t ATP4-)
+ pyruvatel-
In calculations of the n value we used the pK,’ values for the last dissociable group of the magnesium complexes of ATP and ADP. The pKa’ values, at 25’, 0.2 ionic strength, for magnesium ATP and ADP were 4.97 and 5.13, respectively (5). For PEP, pK,’ 5.82 was determined at the same ionic strenght as was used in the determination of PK activity. Using the pKa’ values given above at 7.5, a value of n = 0.96 was calculated. Since n = AH+ taken up per ATP synthesized, the rate of ATP formation can be calculated from the n value obtained at any given pH. 309
@ 1970 by Academic
Preaa, Inc.
310
CARMELI
AND
LIFSHITZ
Reaction Mixture For spectroscopic assay of PK activity, reaction conditions were similar to those used in the method of Bucher (1). In most of the experiments the reaction mixture contained: KCI, 75 mM; Tris-HCI, pH 7.5, 30 mM; MgCl,, 8 mM; PEP, 0.8 mM; NADH, 0.43 mM; ADP, 0.27 mM; lactate dehydrogenase, 20 pg/ml in a total volume of 3 ml at temperature of 23 T 2”. For determination of ADP 0.6 I.U. of PK was used. The reaction was started with the addition of 140 ~1 ADP solution containing Mg,CI, in a ratio of 1:2. For pH measurements the reaction mixture was the same as above except for the omission of NADH and lactate dehydrogenase and for the buffer concentration which was reduced to 1 mM Tris-HCl. HCI, 0.01 IV, was used for determination of the buffer capacity of the reaction mixture. For measurements by titrator the reaction mixture was the same as for pH measurements except for the following variations: in the determination of ADP and PEP Tris buffer was omitted and 0.01 N H.,SO, was used for titration; in the assay of PK activity the concentrations of PEP and ADP were doubled and 0.02 N H&JO4 was used for titration. All acid solutions were standardized against a concentrated volumetric HCl solution purchased from British Drug Houses, Ltd. Apparatus pH changes were measured in a 5 ml cylindrical glass cuvet. The solution was rapidly stirred by a small magnetic stirrer. A Radiometer KG2026C pH electrode was used for pH measurements. The electrode was connected to a Radiometer model 26 pH meter and the recording was done by a Sargent SRG recorder equipped with an auxiliary zero suppression device. Titration was performed by a Radiometer titrator equipped with a pH meter model 26, a titrator model II, a titrograph, and a KG2026C pH electrode. A proportional band of 0.2 was selected, with a syringe buret motor speed of 20 rpm. Spectroscopic measurements were carried out by a Hitachi PerkinElmer model 124 double-beam recording spectrophotometer. RESULTS
AND
DISCUSSION
A recording of the pH changes during PK activity is given in Figure 1. On the addition of ADP aa increase in pH was recorded. It was assumed that the rate of the pH change depended on the rate of PK activity and that the end of the reaction was indicated by the end of the increase
PH
DETERMINATION
OF
PYRUVATE
311
KINASE
- 60 t]’ HCI
0.01 N
FIQ. 1. Recording of pH changes during PK activity with (A) PK present in the medium and (B) PK omitted from the medium. Experimental conditions as described under “Methods.” The pH meter output was 10 mV per 1 unit pH and the recorder was set on 5 mV giving a full scale recording of 0.5 unit pH.
in pH. With limiting amounts of ADP there was almost complete phosphorylation of ADP to ATP at the end of the reaction. Similar changes in pH (not shown here) were recorded when the reaction was started with the addition of PK or PEP instead of ADP, thus indicating that the changes in pH were due to PK activity. The buffer capacity was determined by titration with HCl. Determination of the buffer capacity of the reaction mixture over the same pH range which changed during the reaction ensured accurate results. The initial rate of PK activity was determined from the linear part of the slope at the beginning of the recording. The amount of ADP was determined from the extent of the pH change (the difference between the pH at the beginning and at the end of the reaction). There was a linear relation between the amount of ADP added and the amount measured by this method between 0.1 and 0.7 pmole ADP (Fig. 2). By reducing the buffer concentration and reducing the impedance of the recorder to 1 mV, as little as 0.01 pmole ADP could be measured accurately. Similar results were obtained when the ADP content of a given solution was determined by measuring either changes in pH or titration of hydroxyl ion by the spectroscopic method (Fig, 2). The method revealed a linear relation between the changes in enzyme concentration and the changes in the activity measured (not shown). By reducing the buffer concentration and by increasing the recorder sensitivity as little as 0.03 I.U. of PK activity could be measured. The rate of the change in pH during PK activity was assumed to reflect
312
CARMELI
AND
ADP
LIFSHITZ
pmoles
addedxl0)
FAG. 2. Determination of ADP by three different methods: (0) recording of pH changes, (A) titration with a titrator. ditions so described under “Methods.”
-(0) spectroscopic, Experimental con-
the actual kinetics of the activity because hydroxyl ions are one of the products of the reaction. The quick addition o.f the substrate, the rapid stirring of the solution, and the immediate recording of changes in pH promised reliable results. Recording of pH was found to be 20-fold more sensitive than direct titration both in the measurement of PK activity and in the determination of ADP. SUMMARY
Pyruvate kinase activity was determined by recording changes in pH and by recording titration of the hydroxyl ions released during the reaction. The same methods were also employed for determination of ADP and of phosphoenolpyruvate. The results obtained in the determination of pyruvate kinase activity by following the pH changes and by titration were compared to those obtained by the spectroscopic method using lactate dehydrogenase and NADH. REFERENCES 1. BUCXER, T, in “Methods in Enzymology” (9. P. Colowick and N. 0. Kaplan, eds.), Vol. 1, p. 435. Academic Press, New York, 1955. 2. NISHIMURA, M., ITO, T., AND CHANCE, B., Biochim. Biophys. Acta 59, 177 (1962). 3. KAYNE, F. J., AND SUELTER, C. H., J. Am. Chem. Sot. 87, 897 (1956). 4. MELCHIOR, J. B., Biochemistry 4, 1518 (1965). 5. SMITH, R. M., AND ALBEETY, R. A., .I. Am. Chem. Sac. 78, 2376 (1956).