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New assay for tyrosine aminotransferase with the amino acid analyzer
ANALYTICAL
BIOCHEMISTRY
New
kt,
106110
(1971)
Assay for Tyrosine Aminotransferase with the Amino Acid Analyzer E. A. LANE
Department
of Biochemistry,
AND
C. MAVRIDES
University
of Ottawa,
Received March
Ottawa,
Ontario,
Canada
12, 1971
Most assay methods for tyrosine aminotransferase (n-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) measure the formation of p-hydroxyphenylpyruvic acid (pHPP). The estimation of pHPP is based on the formation of colored products (l-3)) the enol borate-tautomerase reaction (4), the formation of p-hydroxybenzaldehyde from pHPP (5), or the counting of radioactivity derived from the isotopic substrate n-tyrosine (6-8). A recent method is based on the chromatographic separation on cation-exchange paper of 14C-n-glutamic acid formed from W-2-0x0glutaric acid (9). In recent studies in our laboratory the assay of tyrosine aminotransferase by the estimation of the glutamic acid formed was desirable. For this reason we developed a reliable sensitive method in which n-glutamic acid is separated and estimated with the amino acid analyzer. EXPERIMENT’AL
PROCEDURE
Materials. The enzyme preparation was obtained by homogenizing about 30 mg of fresh rat liver with 1.0 ml of 0.1 M sodium phosphate buffer, pH 7.6, in a hand-operated Ten Broeck homogenizer. The homogenate was centrifuged at 27,000g for 15 min and the clear supernatant was used as source of the enzyme. n-Tyrosine and 2-oxoglutaric acid were obtained from Nutritional Biochemicals, diethyldithiocarbamic acid from Eastman Organic Chemicals, and pyridoxal phosphate from Sigma Chemical Company. Assay. The assay mixture in a volume of 2.4 ml contained the following in pmoles, unless otherwise specified: sodium phosphate buffer, pH 7.6, 150; n-tyrosine 8.3; 2-oxoglutaric acid 80.0; diethyldithiocarbamic acid 7.5; pyridoxal phosphate 0.1; and enzyme preparation 0.1 ml. The reaction mixture without the enzyme was preincubated for 20 min at 37°C to solubilize tyrosine and the reaction was started by adding the enzyme. The complete assay mixture was incubated for another 30 min and the reaction was stopped with 0.6 ml of 30% trichloroacetic acid. A blank was 106
ASSAY
OF
TYROSINE
AMIiVOTRANSFERASE
107
prepared by adding trichloroacetic acid just before addition of the enzyme. The precipitated protein was removed by centrifugation and 1.0 ml of the supernatant was used for the separation and estimation of n-glutamic acid. One enzyme unit is the amount of enzyme required to convert 1.0 pmole of 2-oxoglutarate to 1.0 pmole of L-glutamate in 1 min at 37°C. Protein was determined by an automated biuret method (10). Estimation of L-glutamic acid. r,-Glutamic acid was estimated with a Beckman amino acid analyzer, model 1208. A 15 cm column packed with a cation-exchange resin (Aminex A-5, Bio-Rad Laboratories) was used for the separation of glutamic acid from the other components of the reaction mixture. The column was surrounded by a water jacket maintained at 57°C. 1 ml of the deproteinized assay mixture was pipetted onto the column and forced through the resin with air (15 psi). Elution was carried out with 0.2 N sodium citrate buffer, pH 3.28, at a flow rate of 34 ml/ hr. The column effluent was allowed to mix completely in a coil with ninhydrin solution (2%) which was being pumped from a reservoir at a flow rate of 34 ml/hr. The resulting mixture flowed through a coil immersed in a boiling water bath, then through a flow cell, and the absorbance of the purple color in the cell was measured at 570 mp. The calorimetric response was linear with the concentration of glutamic acid in the range of absorbance used for the assays. Under our conditions the glutamic acid was detected in the Aow cell 46.5 min after the start of the pumps. Glutamic acid from assay mixtures was identified by the appearance of a peak at this time which was absent in the blank. The constant for glutamic acid Kglu was calculated by the following relation: K
= peak height X peak width at $ peak height pmoles glutamic acid 1 ml of standard solution containing 0.25 pmole of glutamic acid, passed through the column under the conditions described above, resulted in: peak height = 0.624 OD unit, peak width = 15.2 marks in recorder. Hence K glu = 37.94. By knowing this constant and determining the peak height and peak width, the number of pmoles of glutamic acid in 1.0 ml of assay mixture could be calculated. du
RESULTS
AND
DISCUSSION
Figure 1 shows the elution profiles from a blank and sample tube. The sensitivity of the method allows the detection of glutamic acid in the blank originating in the Iiver extract and possibly in the tyrosine used as substrate in the assay. This blank value was always subtracted from the sample value. The progress curve is shown in Fig. 2. The reaction is linear up to 30
108
LANE
0
10
20
30
AND
40 50 ELUTION
MAVRIDES
TIME
10 20 (min)
30
40
50
FIG. 1. Elution profiles in amino acid analyzer of a blank and an experimental assay mixture for tyrosine aminotransferase. Protein present 0.26 mg.
min and this length of time was routinely used for the assays. Under these conditions the reaction was linear up to the highest amount of the liver extract used in this study corresponding to 0.028 unit of enzyme (Fig. 3). The assay is highly specific since it is based on the complete chromatographic separation of the reaction product and its subsequent estimation by the ninhydrin reaction. The sensitivity of the method is a function of the flow rate, the type of flow cells, and the sensitivity of the instrument itself. Under our conditions 0.025 pmole of n-glutamic acid could be easily estimated and the method is at least as sensitive as the widely used enol borate-tautomerase method. Inherent in the method is the possibility of assaying the enzyme with the reverse reaction in which n-tyrosine would be estimated in the amino acid analyzer. This adds versatility lacking from the assay methods based on the estimation of p-
0
15
30 TIME(min)
45
60
FIQ. 2. Progress curve of tyrosine aminotransferase assay relating tamic acid formed with reaction time. Protein present 0.55 mg.
amount of glu-
ASSAY
OF
TYROSINE
0
1.0 PROTEIN
109
AMINOTRANSFERASE
IN
2.0 ASSAY,
3.0 mg
FIG. 3. Linearity of L-glutamic acid produced versus tyrosine aminotransferase amount. One enzyme unit is amount of enzyme required to convert 1.0 pmole a-oxoglutarate to 1.0 pmole of L-glutamate in 1 min at 37°C.
of
hydroxyphenylpyruvic acid. The possibility for assaying with both the forward and the reverse reaction might be useful in the calculation of the equilibrium constant and related parameters for the purified enzyme from various organisms in comparative studies. An added advantage is that the use of radioactive substrates and the subsequent task of quantitating the radioactivity of the product from paper chromatography are avoided. The relatively long elution time (46.5 min) limits the number of samples that can be handled but this is an inherent disadvantage of all chromatographic procedures. The inhibition of tyrosine aminotransferase by p-hydroxyphenylpyruvic acid, recently reported by Rosenberg and Litwack (11)) who used a radioactive method to study the effect, was detected by our method also, as shown in Table 1. The inhibition would be impossible to detect with the methods which measure pHPP itself formed in the reaction.
Inhibition
pHPP
of Rat
final
Liver
Tyrosine
concentration, 2 x
10-4
1 x
10-z
2 x
10-s
4 x
10-a
TABLE 1 Aminotransferase Acid (pHPP)
M
by p-Hydroxyphenylpyruvic
Per
cent inhibition 14 46 71 87
110
LANE
AND
MAVRIDES
SUMMARY
A new assay method for tyrosine aminotransferase is described based on the automatic separation and estimation of the product L-glutamic acid in an amino acid analyzer. The method is highly specific and offers the possibility of assaying the enzyme with the reverse reaction as well. The inhibition of the enzyme by one of its substrates, p-hydroxyphenylpyruvic acid, was detected by the new method. ACKNOWLEDGMENTS This study was supported by a grant of the Medical Research Council of Canada. We wish to thank Dr. N. L. Benoiton of our department for making available to us the amino acid analyzer and Mr. J. Tong for instructions on its use. REFERENCES 1. 2. 3. 4.
BRIGGS, A. P., J. Bid. ,Chem. 51, 453 (1922). CANELLAKIS, 2. N., AND COHEN, P. P., J. Biol. Chem. 222, 53 (1956). SCHEPARTZ,B., Anal. B&hem. 30, 443 (1969). LIN, E. C. C., PITT, B. M., GIVEN, M., AND KNOX, W. E., J. Biol. Chem. 668
5. 6. 7. 8.
(1958).
DIAMONDSTONE, T. I., Anal. Biochem. 16,395 (1966). WEINSTEIN, A., MEDES, G., AND LITWACK, G., And. Biochem. 21, 86 (1967). LITWACK, G., AND SQUIRES, J. M., Anal. Biochem. 24, 438 (1968). WURTMAN, R. J., AND LARIN, F., Biochem. Pharmacol. 17, 817 (1968). 9. GABAY, S., AND GEORGE,H., Anal. Biochem. 21, 111 (1967). 10. LANE, E. A., AND MAVRIDES, C., Anal. Biochelm. 27,363 (1969). 11. ROSENBERG,J. S., AND LITWACK, G., J. BioZ. Chem. 245, 5677 (1970).
233,
BIOCHEMISTRY
New
kt,
106110
(1971)
Assay for Tyrosine Aminotransferase with the Amino Acid Analyzer E. A. LANE
Department
of Biochemistry,
AND
C. MAVRIDES
University
of Ottawa,
Received March
Ottawa,
Ontario,
Canada
12, 1971
Most assay methods for tyrosine aminotransferase (n-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) measure the formation of p-hydroxyphenylpyruvic acid (pHPP). The estimation of pHPP is based on the formation of colored products (l-3)) the enol borate-tautomerase reaction (4), the formation of p-hydroxybenzaldehyde from pHPP (5), or the counting of radioactivity derived from the isotopic substrate n-tyrosine (6-8). A recent method is based on the chromatographic separation on cation-exchange paper of 14C-n-glutamic acid formed from W-2-0x0glutaric acid (9). In recent studies in our laboratory the assay of tyrosine aminotransferase by the estimation of the glutamic acid formed was desirable. For this reason we developed a reliable sensitive method in which n-glutamic acid is separated and estimated with the amino acid analyzer. EXPERIMENT’AL
PROCEDURE
Materials. The enzyme preparation was obtained by homogenizing about 30 mg of fresh rat liver with 1.0 ml of 0.1 M sodium phosphate buffer, pH 7.6, in a hand-operated Ten Broeck homogenizer. The homogenate was centrifuged at 27,000g for 15 min and the clear supernatant was used as source of the enzyme. n-Tyrosine and 2-oxoglutaric acid were obtained from Nutritional Biochemicals, diethyldithiocarbamic acid from Eastman Organic Chemicals, and pyridoxal phosphate from Sigma Chemical Company. Assay. The assay mixture in a volume of 2.4 ml contained the following in pmoles, unless otherwise specified: sodium phosphate buffer, pH 7.6, 150; n-tyrosine 8.3; 2-oxoglutaric acid 80.0; diethyldithiocarbamic acid 7.5; pyridoxal phosphate 0.1; and enzyme preparation 0.1 ml. The reaction mixture without the enzyme was preincubated for 20 min at 37°C to solubilize tyrosine and the reaction was started by adding the enzyme. The complete assay mixture was incubated for another 30 min and the reaction was stopped with 0.6 ml of 30% trichloroacetic acid. A blank was 106
ASSAY
OF
TYROSINE
AMIiVOTRANSFERASE
107
prepared by adding trichloroacetic acid just before addition of the enzyme. The precipitated protein was removed by centrifugation and 1.0 ml of the supernatant was used for the separation and estimation of n-glutamic acid. One enzyme unit is the amount of enzyme required to convert 1.0 pmole of 2-oxoglutarate to 1.0 pmole of L-glutamate in 1 min at 37°C. Protein was determined by an automated biuret method (10). Estimation of L-glutamic acid. r,-Glutamic acid was estimated with a Beckman amino acid analyzer, model 1208. A 15 cm column packed with a cation-exchange resin (Aminex A-5, Bio-Rad Laboratories) was used for the separation of glutamic acid from the other components of the reaction mixture. The column was surrounded by a water jacket maintained at 57°C. 1 ml of the deproteinized assay mixture was pipetted onto the column and forced through the resin with air (15 psi). Elution was carried out with 0.2 N sodium citrate buffer, pH 3.28, at a flow rate of 34 ml/ hr. The column effluent was allowed to mix completely in a coil with ninhydrin solution (2%) which was being pumped from a reservoir at a flow rate of 34 ml/hr. The resulting mixture flowed through a coil immersed in a boiling water bath, then through a flow cell, and the absorbance of the purple color in the cell was measured at 570 mp. The calorimetric response was linear with the concentration of glutamic acid in the range of absorbance used for the assays. Under our conditions the glutamic acid was detected in the Aow cell 46.5 min after the start of the pumps. Glutamic acid from assay mixtures was identified by the appearance of a peak at this time which was absent in the blank. The constant for glutamic acid Kglu was calculated by the following relation: K
= peak height X peak width at $ peak height pmoles glutamic acid 1 ml of standard solution containing 0.25 pmole of glutamic acid, passed through the column under the conditions described above, resulted in: peak height = 0.624 OD unit, peak width = 15.2 marks in recorder. Hence K glu = 37.94. By knowing this constant and determining the peak height and peak width, the number of pmoles of glutamic acid in 1.0 ml of assay mixture could be calculated. du
RESULTS
AND
DISCUSSION
Figure 1 shows the elution profiles from a blank and sample tube. The sensitivity of the method allows the detection of glutamic acid in the blank originating in the Iiver extract and possibly in the tyrosine used as substrate in the assay. This blank value was always subtracted from the sample value. The progress curve is shown in Fig. 2. The reaction is linear up to 30
108
LANE
0
10
20
30
AND
40 50 ELUTION
MAVRIDES
TIME
10 20 (min)
30
40
50
FIG. 1. Elution profiles in amino acid analyzer of a blank and an experimental assay mixture for tyrosine aminotransferase. Protein present 0.26 mg.
min and this length of time was routinely used for the assays. Under these conditions the reaction was linear up to the highest amount of the liver extract used in this study corresponding to 0.028 unit of enzyme (Fig. 3). The assay is highly specific since it is based on the complete chromatographic separation of the reaction product and its subsequent estimation by the ninhydrin reaction. The sensitivity of the method is a function of the flow rate, the type of flow cells, and the sensitivity of the instrument itself. Under our conditions 0.025 pmole of n-glutamic acid could be easily estimated and the method is at least as sensitive as the widely used enol borate-tautomerase method. Inherent in the method is the possibility of assaying the enzyme with the reverse reaction in which n-tyrosine would be estimated in the amino acid analyzer. This adds versatility lacking from the assay methods based on the estimation of p-
0
15
30 TIME(min)
45
60
FIQ. 2. Progress curve of tyrosine aminotransferase assay relating tamic acid formed with reaction time. Protein present 0.55 mg.
amount of glu-
ASSAY
OF
TYROSINE
0
1.0 PROTEIN
109
AMINOTRANSFERASE
IN
2.0 ASSAY,
3.0 mg
FIG. 3. Linearity of L-glutamic acid produced versus tyrosine aminotransferase amount. One enzyme unit is amount of enzyme required to convert 1.0 pmole a-oxoglutarate to 1.0 pmole of L-glutamate in 1 min at 37°C.
of
hydroxyphenylpyruvic acid. The possibility for assaying with both the forward and the reverse reaction might be useful in the calculation of the equilibrium constant and related parameters for the purified enzyme from various organisms in comparative studies. An added advantage is that the use of radioactive substrates and the subsequent task of quantitating the radioactivity of the product from paper chromatography are avoided. The relatively long elution time (46.5 min) limits the number of samples that can be handled but this is an inherent disadvantage of all chromatographic procedures. The inhibition of tyrosine aminotransferase by p-hydroxyphenylpyruvic acid, recently reported by Rosenberg and Litwack (11)) who used a radioactive method to study the effect, was detected by our method also, as shown in Table 1. The inhibition would be impossible to detect with the methods which measure pHPP itself formed in the reaction.
Inhibition
pHPP
of Rat
final
Liver
Tyrosine
concentration, 2 x
10-4
1 x
10-z
2 x
10-s
4 x
10-a
TABLE 1 Aminotransferase Acid (pHPP)
M
by p-Hydroxyphenylpyruvic
Per
cent inhibition 14 46 71 87
110
LANE
AND
MAVRIDES
SUMMARY
A new assay method for tyrosine aminotransferase is described based on the automatic separation and estimation of the product L-glutamic acid in an amino acid analyzer. The method is highly specific and offers the possibility of assaying the enzyme with the reverse reaction as well. The inhibition of the enzyme by one of its substrates, p-hydroxyphenylpyruvic acid, was detected by the new method. ACKNOWLEDGMENTS This study was supported by a grant of the Medical Research Council of Canada. We wish to thank Dr. N. L. Benoiton of our department for making available to us the amino acid analyzer and Mr. J. Tong for instructions on its use. REFERENCES 1. 2. 3. 4.
BRIGGS, A. P., J. Bid. ,Chem. 51, 453 (1922). CANELLAKIS, 2. N., AND COHEN, P. P., J. Biol. Chem. 222, 53 (1956). SCHEPARTZ,B., Anal. B&hem. 30, 443 (1969). LIN, E. C. C., PITT, B. M., GIVEN, M., AND KNOX, W. E., J. Biol. Chem. 668
5. 6. 7. 8.
(1958).
DIAMONDSTONE, T. I., Anal. Biochem. 16,395 (1966). WEINSTEIN, A., MEDES, G., AND LITWACK, G., And. Biochem. 21, 86 (1967). LITWACK, G., AND SQUIRES, J. M., Anal. Biochem. 24, 438 (1968). WURTMAN, R. J., AND LARIN, F., Biochem. Pharmacol. 17, 817 (1968). 9. GABAY, S., AND GEORGE,H., Anal. Biochem. 21, 111 (1967). 10. LANE, E. A., AND MAVRIDES, C., Anal. Biochelm. 27,363 (1969). 11. ROSENBERG,J. S., AND LITWACK, G., J. BioZ. Chem. 245, 5677 (1970).
233,