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Assay of ornithine aminotransferase by high-performance liquid chromatography
ANALYTICAL
BIOCHEMISTRY
90.
41-46 (1978)
Assay of Ornithine Aminotransferase by HighPerformance Liquid Chromatography JAMES J. O'DONNELL,ROBERT P. SANDMAN,AND Department
of Ophthalmology, U-490, San Francisco,
University Calijbrnia
of California, 94143
SUSAN R. MARTIN San Francisco,
Received December 19, 1977 Omithine aminotransferase has been measured previously with a spectrophotometric assay and with a radioactive assay. We report here an isocratic reverse phase high-performance liquid chromatography assay which measures A’pyrroline-S-carboxylic acid, the reaction product. This assay offers the advantages of sensitivity and convenience.
Omithine aminotransferase (L-ornithine:2-oxoacid aminotransferase EC 2.6.1.13) is a mitochondrial matrix enzyme that catalyzes the interaction of L-ornithine and a-ketoglutarate to produce glutamic-y-semialdehyde and glutamate (1,2). Spontaneous cyclization converts glutamicy-semialdehyde to Al-pyrroline-5-carboxylic acid (PX),’ a proline precursor. Ornithine aminotransferase (OTA) has been assayed with a spectrophotometric method (1) and with a radioactive method (3,4). We now report a high-performance liquid chromatography (hplc) assay for OTA. The principle of the assay is that PX, the reaction product, reacts with o-aminobenzaldehyde (OAB), and the resultant dihydroquinozolinium compound is separated by hplc and detected by its absorbance at 254 nm. METHODS
The enzyme assay is a modification of the method of Strecker (1). The liver from an adult AC1 female rat was homogenized in a 20% (w/v) solution containing 0.1 M KPO, (ph 7.4) buffer with 0.25 M sucrose and 4 pg/ml pyridoxal phosphate at 4°C. The supernatant from a 15-min. IOOO-rpm centrifugation was stored frozen overnight at -20°C. For enzyme assay, this homogenate was added to start the reaction in a freshly prepared mixture containing 35 mM L-ornithine (Sigma), 3.75 mM r Abbreviations used: PSC,A’-pyrroline-Scarboxylic acid; OTA, omithine aminotransferase; hplc, high-performance liquid chromatography; DHQ, dihydroquinozolinium; and OAB, o-aminobenzaldehyde. 41
0003-2697/78/0901-0041$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
42
O’DONNELL.
SANDMAN.
AND
MARTIN
cy-ketoglutarate (Sigma), 50 mM KPO, (ph 7.4), and 4 pgiml pyridoxal phosphate in a total volume of 2 ml at 37°C. The reaction was terminated by the addition of 1 ml of 3 N HCl containing 7.5 mg/ml o-aminobenzaldehyde (Sigma), and the samples were centrifuged at 3000 rpm to remove the precipitated protein. HCl was used to precipitate protein instead of trichloroacetic acid because trichloroacetic acid causes significant interference with the chromatogram at 254 nm. Blanks were prepared by adding the homogenate after the HCl. The absorbance of the dihydroquinozolinium formed by the P5C and the OAB was determined with a Zeiss PMQ2 spectrophotometer at 440 nm, and the recently reported (5) millimolar extinction coefficient of 2.590 at 440 nm was used to calculate concentration. Activity is expressed as micromoles of P5C formed per hour per milligram of liver wet weight. Isocratic reverse phase hplc was accomplished with a LiChrosorb C,,, IO-pm, 4.6 x 250-mm column (Upchurch Scientific, 2243 Old Middlefield Way, Mountain View, California 94043) and an Altex model 310 chromatograph (Upchurch Scientific) with 254-nm detection (Fig. 1). The detector cell had a lo-mm path length and 8-~1 volume. The solvent system of 1 part spectral grade methanol:2 parts Hz0 was degassed by filtering it with suction through a 0.45~pm Millipore filter and was pumped at 1.5 ml/ min at room temperature. Ten-microliter samples were applied to the column. P5C was freshly synthesized by periodate oxidation of DL-hydroxylysine (5) and reacted with OAB to form dihydroquinazolinium, which agreed with the reported spectral absorbance (6) determined with a Zeiss PMQ,
MINUTES FIG. 1. Isocratic reverse-phase hplc showing separation and detection of dihydroquinozolinium (DHQ) and
ORNITHINE
43
AMINOTRANSFERASE
I50 -
200
I 240
/ 280
I 320
I 360
I 400
I 440
WAVELENGTH
FIG. 2. Spectrum of dihydroquinozolinium quinozolinium was collected as a fraction
I 480
I 520
I 560
I 600
I 640
I 680
nm
in 1 part methanol:2 from the hplc.
parts
H,O.
The dihydro-
spectrophotometer (Fig. 2). This authentic dihydroquinozolinium was used to determine the above chromatographic retention time and to prepare the standard curve of concentration and peak height (Fig. 3) used to calculate the P5C in the hplc. RESULTS
The activity determined with hplc was linear with time and with added homogenate (Fig 5). P5C concentrations the spectrophotometric method and with the hplc method (Fig. 6). Enzyme activity with the spectrophotometric
to 60 min (Fig. 4) determined with were comparable method was 71.2
l"r
HPLC OPTICAL DENSITY AT 254 nm ( xIO-~)
FIG. 3. Standard curve of hplc absorbance and PSC concentration. Least squares linear regression: (PSC M/L) = 2.56 x IO-” optical density + I .78 hplc optical density, r = 0.997. Peak heights rather than peak area were used. The points are experimental and the curve is a calculated least squares plot.
44
O’DONNELL,
SANDMAN,
15
AND MARTIN
30
45
60
MINUTES
FIG. 4. OTA activity as determined by hplc (see text) plotted against time. The reaction is linear for 60 min.
k 10.9 (SD) and with the hplc method was 71.8 k 6.0 (SD) prnol of PSC formed/hr/mg of liver wet weight. DISCUSSION
This hplc determination of OTA activity offers advantages over the spectrophotometric method in that small samples with low activity can be assayed. 3.9 nmoles of the dihydroquinozolinium in solution gives an optical density of 0.010 at 440 nm in a l-ml l-cm light path cuvette in the spectrophotometer. This is the lower limit of reliable optical density on most spectrophotometers. Microcuvettes can be obtained to decrease the amount of material detected by a factor of 10. The hplc system described 2.00 E H h 0
II) a
0 1.00
s P =I.
I!!
50 100
ADDED
200
200
HOMOGENATE (Lxlo-6)
FIG. 5. OTA activity determined by hplc plotted against added homogenate. The reaction is linear with homogenate.
ORNITHINE
45
AMINOTRANSFERASE
HPLC
ASSAY
tP5C
m/Lxd1
FIG. 6. OTA activity determined with the spectrophotometric assay (1) plotted against activity determined with hplc. Least squares linear regression: spectrophotometric activity (P5C M/L) = 6.47 x 10m6 (P5C M/L) + 1.02 hplc activity (PX M/L). r = 0.977. The points are experimental and the curve is a calculated least squares plot.
here can detect 25 pmol of the dihydroquinozolinium with the sample peak twice the short term baseline noise. Much of this increased sensitivity is attributable to the small aliquot (10 ~1) used. The radioactive assay can detect P5C at a sensitivity similar to that found with the hplc method. The radioactive assay, however, is time consuming and depends on the radioactive labeling specificity and stability of the ornithine used. Kinetic studies on low activity samples are difficult with the radioactive assay because of the difficulty in obtaining high specific activity ornithine. In addition, one of the described radioactive assays (4) notes an inhibitor in the radioactive ornithine. A 254-nm detection is used here to take advantage of the 2%nm emission line of low-pressure mercury lamps and the high absorbance of dihydroquinozolinium at this wavelength. Figure 2 shows that a 300-nm detection would also work well. One should be cautious, however, to use a narrow bandpass detector at 300 nm to ensure a linear response in accord with Beer’s law. The lower absorbance at 440 nm was used in the original spectrophotometric method because the dihydroquinozolinium was not separated from interfering substances in the assay at lower wavelengths. The method will be especially useful in investigations of cultured cells where limited material is available. The largest aliquot that can be placed on the described hplc system is 20 ~1. The response is linear from 1 to 20 ~1. High performance liquid chromatography detection of P5C may be useful for assay of other enzymes involved in PSC metabolism such as P5C dehydrogenase, PSC reductase, and proline oxidase. ACKNOWLEDGMENTS This work was supported by a Basil O’Connor Award Foundation-March of Dimes and by the National Eye
to J. O’Donnell Institute, Grant
from the National EY 01786-02.
46
O’DONNELL,
SANDMAN,
AND MARTIN
REFERENCES 1. 2. 3. 4.
Strecker, H. J. (1965)5. Biol. Chem. 240, 1225-1230. Peraino, C., Bunville, L. G., and Tatimisian, T. (1969) J. Bid. Chem. 244, 2241-2249. Phang, J. M., Downing, S. J., and Valle. D. (1973) And. Eiochem. 55, 272-277. Trijbels. J. M. F.. Sengers. R. C. A., Bakkeren. J. A. J. M.. DeKort, A. F. M., and Deutman. A. F. (1977) C/in. Chim. Acta 79, 371-377. 5. Mezl, V. A.. and Knox, W. E. (1976) Anal. Biochem. 74, 430-440. 6. Strecker. H. J. (1960) J. Bid. Chrm. 235, 2045-2050.
BIOCHEMISTRY
90.
41-46 (1978)
Assay of Ornithine Aminotransferase by HighPerformance Liquid Chromatography JAMES J. O'DONNELL,ROBERT P. SANDMAN,AND Department
of Ophthalmology, U-490, San Francisco,
University Calijbrnia
of California, 94143
SUSAN R. MARTIN San Francisco,
Received December 19, 1977 Omithine aminotransferase has been measured previously with a spectrophotometric assay and with a radioactive assay. We report here an isocratic reverse phase high-performance liquid chromatography assay which measures A’pyrroline-S-carboxylic acid, the reaction product. This assay offers the advantages of sensitivity and convenience.
Omithine aminotransferase (L-ornithine:2-oxoacid aminotransferase EC 2.6.1.13) is a mitochondrial matrix enzyme that catalyzes the interaction of L-ornithine and a-ketoglutarate to produce glutamic-y-semialdehyde and glutamate (1,2). Spontaneous cyclization converts glutamicy-semialdehyde to Al-pyrroline-5-carboxylic acid (PX),’ a proline precursor. Ornithine aminotransferase (OTA) has been assayed with a spectrophotometric method (1) and with a radioactive method (3,4). We now report a high-performance liquid chromatography (hplc) assay for OTA. The principle of the assay is that PX, the reaction product, reacts with o-aminobenzaldehyde (OAB), and the resultant dihydroquinozolinium compound is separated by hplc and detected by its absorbance at 254 nm. METHODS
The enzyme assay is a modification of the method of Strecker (1). The liver from an adult AC1 female rat was homogenized in a 20% (w/v) solution containing 0.1 M KPO, (ph 7.4) buffer with 0.25 M sucrose and 4 pg/ml pyridoxal phosphate at 4°C. The supernatant from a 15-min. IOOO-rpm centrifugation was stored frozen overnight at -20°C. For enzyme assay, this homogenate was added to start the reaction in a freshly prepared mixture containing 35 mM L-ornithine (Sigma), 3.75 mM r Abbreviations used: PSC,A’-pyrroline-Scarboxylic acid; OTA, omithine aminotransferase; hplc, high-performance liquid chromatography; DHQ, dihydroquinozolinium; and OAB, o-aminobenzaldehyde. 41
0003-2697/78/0901-0041$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
42
O’DONNELL.
SANDMAN.
AND
MARTIN
cy-ketoglutarate (Sigma), 50 mM KPO, (ph 7.4), and 4 pgiml pyridoxal phosphate in a total volume of 2 ml at 37°C. The reaction was terminated by the addition of 1 ml of 3 N HCl containing 7.5 mg/ml o-aminobenzaldehyde (Sigma), and the samples were centrifuged at 3000 rpm to remove the precipitated protein. HCl was used to precipitate protein instead of trichloroacetic acid because trichloroacetic acid causes significant interference with the chromatogram at 254 nm. Blanks were prepared by adding the homogenate after the HCl. The absorbance of the dihydroquinozolinium formed by the P5C and the OAB was determined with a Zeiss PMQ2 spectrophotometer at 440 nm, and the recently reported (5) millimolar extinction coefficient of 2.590 at 440 nm was used to calculate concentration. Activity is expressed as micromoles of P5C formed per hour per milligram of liver wet weight. Isocratic reverse phase hplc was accomplished with a LiChrosorb C,,, IO-pm, 4.6 x 250-mm column (Upchurch Scientific, 2243 Old Middlefield Way, Mountain View, California 94043) and an Altex model 310 chromatograph (Upchurch Scientific) with 254-nm detection (Fig. 1). The detector cell had a lo-mm path length and 8-~1 volume. The solvent system of 1 part spectral grade methanol:2 parts Hz0 was degassed by filtering it with suction through a 0.45~pm Millipore filter and was pumped at 1.5 ml/ min at room temperature. Ten-microliter samples were applied to the column. P5C was freshly synthesized by periodate oxidation of DL-hydroxylysine (5) and reacted with OAB to form dihydroquinazolinium, which agreed with the reported spectral absorbance (6) determined with a Zeiss PMQ,
MINUTES FIG. 1. Isocratic reverse-phase hplc showing separation and detection of dihydroquinozolinium (DHQ) and
ORNITHINE
43
AMINOTRANSFERASE
I50 -
200
I 240
/ 280
I 320
I 360
I 400
I 440
WAVELENGTH
FIG. 2. Spectrum of dihydroquinozolinium quinozolinium was collected as a fraction
I 480
I 520
I 560
I 600
I 640
I 680
nm
in 1 part methanol:2 from the hplc.
parts
H,O.
The dihydro-
spectrophotometer (Fig. 2). This authentic dihydroquinozolinium was used to determine the above chromatographic retention time and to prepare the standard curve of concentration and peak height (Fig. 3) used to calculate the P5C in the hplc. RESULTS
The activity determined with hplc was linear with time and with added homogenate (Fig 5). P5C concentrations the spectrophotometric method and with the hplc method (Fig. 6). Enzyme activity with the spectrophotometric
to 60 min (Fig. 4) determined with were comparable method was 71.2
l"r
HPLC OPTICAL DENSITY AT 254 nm ( xIO-~)
FIG. 3. Standard curve of hplc absorbance and PSC concentration. Least squares linear regression: (PSC M/L) = 2.56 x IO-” optical density + I .78 hplc optical density, r = 0.997. Peak heights rather than peak area were used. The points are experimental and the curve is a calculated least squares plot.
44
O’DONNELL,
SANDMAN,
15
AND MARTIN
30
45
60
MINUTES
FIG. 4. OTA activity as determined by hplc (see text) plotted against time. The reaction is linear for 60 min.
k 10.9 (SD) and with the hplc method was 71.8 k 6.0 (SD) prnol of PSC formed/hr/mg of liver wet weight. DISCUSSION
This hplc determination of OTA activity offers advantages over the spectrophotometric method in that small samples with low activity can be assayed. 3.9 nmoles of the dihydroquinozolinium in solution gives an optical density of 0.010 at 440 nm in a l-ml l-cm light path cuvette in the spectrophotometer. This is the lower limit of reliable optical density on most spectrophotometers. Microcuvettes can be obtained to decrease the amount of material detected by a factor of 10. The hplc system described 2.00 E H h 0
II) a
0 1.00
s P =I.
I!!
50 100
ADDED
200
200
HOMOGENATE (Lxlo-6)
FIG. 5. OTA activity determined by hplc plotted against added homogenate. The reaction is linear with homogenate.
ORNITHINE
45
AMINOTRANSFERASE
HPLC
ASSAY
tP5C
m/Lxd1
FIG. 6. OTA activity determined with the spectrophotometric assay (1) plotted against activity determined with hplc. Least squares linear regression: spectrophotometric activity (P5C M/L) = 6.47 x 10m6 (P5C M/L) + 1.02 hplc activity (PX M/L). r = 0.977. The points are experimental and the curve is a calculated least squares plot.
here can detect 25 pmol of the dihydroquinozolinium with the sample peak twice the short term baseline noise. Much of this increased sensitivity is attributable to the small aliquot (10 ~1) used. The radioactive assay can detect P5C at a sensitivity similar to that found with the hplc method. The radioactive assay, however, is time consuming and depends on the radioactive labeling specificity and stability of the ornithine used. Kinetic studies on low activity samples are difficult with the radioactive assay because of the difficulty in obtaining high specific activity ornithine. In addition, one of the described radioactive assays (4) notes an inhibitor in the radioactive ornithine. A 254-nm detection is used here to take advantage of the 2%nm emission line of low-pressure mercury lamps and the high absorbance of dihydroquinozolinium at this wavelength. Figure 2 shows that a 300-nm detection would also work well. One should be cautious, however, to use a narrow bandpass detector at 300 nm to ensure a linear response in accord with Beer’s law. The lower absorbance at 440 nm was used in the original spectrophotometric method because the dihydroquinozolinium was not separated from interfering substances in the assay at lower wavelengths. The method will be especially useful in investigations of cultured cells where limited material is available. The largest aliquot that can be placed on the described hplc system is 20 ~1. The response is linear from 1 to 20 ~1. High performance liquid chromatography detection of P5C may be useful for assay of other enzymes involved in PSC metabolism such as P5C dehydrogenase, PSC reductase, and proline oxidase. ACKNOWLEDGMENTS This work was supported by a Basil O’Connor Award Foundation-March of Dimes and by the National Eye
to J. O’Donnell Institute, Grant
from the National EY 01786-02.
46
O’DONNELL,
SANDMAN,
AND MARTIN
REFERENCES 1. 2. 3. 4.
Strecker, H. J. (1965)5. Biol. Chem. 240, 1225-1230. Peraino, C., Bunville, L. G., and Tatimisian, T. (1969) J. Bid. Chem. 244, 2241-2249. Phang, J. M., Downing, S. J., and Valle. D. (1973) And. Eiochem. 55, 272-277. Trijbels. J. M. F.. Sengers. R. C. A., Bakkeren. J. A. J. M.. DeKort, A. F. M., and Deutman. A. F. (1977) C/in. Chim. Acta 79, 371-377. 5. Mezl, V. A.. and Knox, W. E. (1976) Anal. Biochem. 74, 430-440. 6. Strecker. H. J. (1960) J. Bid. Chrm. 235, 2045-2050.