electrochemistry

ANALYTICAL

BIOCHEMISTRY

139,468-473

(1984)

Determination of Phenylalanine Hydroxylase by Liquid Chromatography/Electrochemistry

Activity

CRAIG E. LUNTE AND PETER T. KISSINGER Department of Chemistry, Purdue University, West Lafayette, Indiana Received July 18, 1983 An assay method is presented for the determination of phenylalanine hydroxylase activity in biological samples. The procedure is rapid and requires little sample. Multiple components of the enzyme system are determined and therefore serve as internal checks of the assay system. Liquid chromatography/electrochemistry is employed to follow the oxidation of the tetrahydropterin cofactor to the dihydropterin and to follow the formation of tyrosine. The KM and V,,,,, values of both phenylalanine and 6-methyl-5,6,7,8-tetrahydropterin were determined for mouse liver phenylalanine hydroxylase. Determination of the stoichiometry of the reaction showed that 1 mol of dihydropterin and 1 mol of tyrosine are formed per mole of tetrahydropterin that is oxidized. The reaction rate was linear for several minutes and over a wide range of enzyme (protein) concentrations. KEY WORDS: phenylalanine hydroxylase; tetrahydropterin; tyrosine; liquid chromatography; electrochemical detection.

Phenylalanine hydroxylase (EC 1.14.16.1) catalyzes the conversion of phenylalanine to tyrosine. The enzyme requires a reduced pterin as a cofactor; the natural cofactor being 5,6,7,8-tetrahydrobiopterin (1). In the enzymatic reaction, phenylalanine is hydroxylated to form tyrosine, and tetrahydrobiopterin is oxidized to quinonoid dihydrobiopterin. The tetrahydrobiopterin is regenerated by the reduction of the quinonoid dihydrobiopterin by NADH and the enzyme dihydropteridine reductase (EC 1699.7) (2). This system is illustrated in Fig. 1. Several approaches have been taken to the determination of phenylalanine hydroxylase activity. The tyrosine formed has been derivitized and determined either spectrophotometrically (3) or fluorometrically (4). The tyrosine has also been determined fluorometrically following separation by liquid chromatography (5). The enzyme activity has also been determined by using radiolabeled phenylalanine (6). Ayling et al. (7) have described a method of spectrophotometrically following the formation of the dihydropterin. Fi0003-2697184 53.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.

468

nally, a coupled enzyme system has been employed using dihydropteridine reductase/ NADH, with the oxidation of NADH being followed spectrophotometrically (8). We have shown that reduced pterins can readily be detected by liquid chromatography/ electrochemistry (LCEC)’ (9). Using LCEC to quantitate the oxidation of the tetrahydropterin cofactor, a direct determination of phenylalaniae hydroxylase activity is possible without the need of derivatization or supplementary enzyme systems. In addition, the product of phenylalanine hydroxylation, tyrosine, is electroactive and can be simultaneously quantitated. We have developed an LCEC methodology for the determination of phenylalanine hydroxylase activity based on the oxidation of the tetrahydropterin cofactor and on the formation of both the dihydropterin and tyrosine. Recently, a similar method was described using synthetic, non-pterin co’ Abbreviations

used: LCEC,

liquid

chromatography/

electrochemistry; MP&, 6-methyl-5,6,7,8-tetrahydropterin; MPH*, 6-methyl-7,8-dihydropterin.

DETERMINATION MI

TETRAHKJNOPTERIN

WNONOID

DIHYDROPTERIN

OF PHENYLALANINE x-+

NADH

NE

t

7,8-DIHYDROPTERIN

FIG. 1. Phenylalanine hydroxylating system. PAH, phenylalanine hydroxylase; QDHR, quinonoid dihydropterin reductase; NE, nonenzymatic path.

factors to study the intermediate state of the pterin cofactor ( 10). This methodology has been used to determine the phenylalanine hydroxylase activity in mouse liver. The Z& and V,,,,X values of phenylalanine and 6-methyl-5,6,7,8-tetrahydropterin have also been determined for mouse liver phenylalanine hydroxylase. MATERIALS

AND METHODS

Chemicals. All chemicals were reagent grade or better. 6-Methyl-5,6,7,8-tetrahydropterin (MPH4) and 6-methyl-7,8-dihydropterin (MPH*) were purchased from Calbiochem-Behring (La Jolla, Calif.). L-Tyrosine and L-phenylalanine were obtained from Sigma (St. Louis, MO.). Syringic acid and octyl sodium sulfate were purchased from EastmanKodak (Rochester, N. Y.). Apparatus. The chromatographic system employed consisted of an Altex 110 pump with a pulse dampener and a Rheodyne 70 10 injection valve with a 20-~1 sample loop. A Brownlee MPLC RP-18 5-pm (4.6 mm X 10 cm) column was employed. Detection was with an LC4B amperometric detector (Bioanalytical Systems, West Lafayette, Ind.) using a glassy carbon working electrode. A Ag/AgCl electrode was used as the reference. Liquid chromatography. An “ion-pair” reverse-phase chromatographic system was used to achieve separation of the various analytes. The mobile phase was 3 mM octyl sodium sulfate in a 0.1 M sodium phosphate buffer, pH 2.5, with 10% methanol (v/v). The mobile

HYDROXYLASE

ACTIVITY

469

phase was prepared from distilled, deionized water and filtered through a 0.22-pm filter (Millipore, Milford, Mass.). Because the reduced pterins are extremely labile to oxidation, oxygen was removed from the mobile phase by continuous purging with nitrogen and maintaining the mobile phase reservoir at a temperature of 40°C. A flow rate of 2.0 ml/ min was used for all experiments. Sample preparation. Mice were killed by decapitation and their livers removed. The livers were rinsed with 0.1 M sodium phosphate buffer, pH 6.8, to remove blood. The livers were then homogenized in 10 ml of 0.1 M phosphate buffer, pH 6.8. This homogenate was used without further purification as the enzyme source. Protein in the sample was determined by the method of Lowry et al. (11). Bovine serum albumin was used as the standard. Incubation procedure. All solutions were prepared in 0.1 M sodium phosphate buffer, pH 6.8, except for 6-methyl-5,6,7,8-tetrahydropterin which was prepared in deoxygenated 0.0 1 M phosphoric acid. The incubation mixture for determination of phenylalanine hydroxylase activity consisted of 50 pg catalase (1300 units), 1.0 pmol phenylalanine, 0.15 pmol MPH4, 0.17 pmol syringic acid, and the liver homogenate in a final volume of 500 ~1. Prior to the addition of MPH4, the incubation mixture was shaken well in order to saturate the solution with oxygen. The incubation was started by the addition of MPH4 with vortex mixing. After 2 min the reaction was quenched by the addition of 200 ~1 of cold 1.O M HC104. The quenched incubation was centrifuged for 5 min at 15,000g and a 200~~1 aliquot was diluted to 2.0 ml with 0.1 M sodium phosphate buffer, pH 2.5, and 0.1 mM ascorbic acid. This sample was then subjected to analysis by LCEC to determine the MPH4, MPH2, and tyrosine concentrations. In addition to the enzymatic oxidation, MPH4 is nonenzymatically oxidized under the assay conditions. The rate of nonenzymatic oxidation is dependent upon the amount of sample in the incubation. Therefore, a blank

470

LUNTE

AND KISSINGER

must be run for each sample. The blank consisted of the incubation mixture without any phenylalanine added. The use of a blank also eliminates any contributions from endogenous tyrosine. Internal standard. The use of an internal standard reduces variances due to sampling and transfer of solutions during incubation, centrifugation and dilution. Due to favorable chromatographic and electrochemical behavior, syringic acid was chosen as an internal standard. No detectable oxidation of the syringic acid was observed during the time of the incubations. Activity calculations. The response (peak height) of all sample components were normalized to the average response of syringic acid. The concentrations of the components were then determined from standard curves. The blank responses were subtracted from the sample responses and the dilutions accounted for to give concentrations in units of micromoles/milliliter for the original 500~~1 incubation mixture. The activity of phenylalanine hydroxylase was then calculated in terms of nanomoles/minute/milligram of protein.

0

E(volts) FIG. 2. Hydrodynamic voltammograms. (0) MP&, (w) MPHz, (A) syringic acid, (0) tyrosine.

A

PH4

B

T 1

IO nA

PH4 SA

MPH2

L

I

0

2

4

6

0

2

4

6

minutes FIG. 3. Chromatographic separation. (A) blank, (B) sample (activity was 3.36 nmol/min/mg protein). Chromatographic conditions as in text.

RESULTS AND DISCUSSION

Electrochemistry. MPH4, MPH2, tyrosine, and syringic acid can all be oxidized at a glassy carbon electrode at potentials accessible by LCEC. Chromatographically assisted hydrodynamic voltammograms (HDV’s) for these compounds are shown in Fig. 2. It can be seen from the HDV’s that a potential of + 1.2 V versus the Ag/AgCl electrode is on the limiting current plateau of all compounds and is therefore suitable for electrochemical detection. Chromatography. For this methodology to be useful, the chromatographic analysis must be rapid, yet still capable of fully resolving all of the analytically significant compounds. To accomplish this, a “fast” LC system was employed, in other words, a short column with elevated flow rates. Typical chromatograms obtained for a sample and the corresponding blank are shown in Fig. 3. Using “fast” LC approximately 10 samples/h can be analyzed. Initial rate dependence. Under the assay conditions, the reaction was linear for about 3 min (Fig. 4). The use of a 2-min incubation

DETERMINATION

0

I

2

OF PHENYLALANINE

3

4

HYDROXYLASE

V 0

I

471

ACTIVITY

2

3

4

minutes

FIG. 4. Rate of reaction. The data points represent the average value of the amount of MPH, oxidized and the amount of MPH2 and tyrosine formed after subtraction of the blank values. The incubation contained 2.76 mg protein.

FIG. 5. Rate of nonenzymatic oxidation of MPH4. The incubation contained 2.76 mg protein and no phenylalanine.

termination (Table 1). These values compare well with previously reported values (12). Stoichiometry of the reaction. The amount

time for determination of phenylalanine hydroxylase activity ensures remaining in the linear region. The nonenzymatic oxidation of MPH4 remains linear for greater than 4 min (Fig. 5). The short incubation time also ensures that the enzymatic oxidation of MPH4 is the major pathway of oxidation. At longer incubation times a greater portion of the oxidation is due to nonenzymatic pathways and the reproducibility of the analysis decreases. The reaction rate was also a linear function of enzyme (protein) concentration up to 3 mg of protein (Fig. 6). Samples of less than 1 mg of protein can be analyzed by using less MPH4 and a smaller dilution. Determining only the tyrosine can also be useful in the analysis of samples with very low activity. Kinetic parameters. Apparent KM and V,,,,, 1.0 21) 30 4.0 values were determined for MPH4 and pheMG PROTEIN nylalanine from the formation of MPH2 and FIG. 6. Initial rate of reaction dependence on protein tyrosine and from the oxidation of MPH4. concentration. The data points represent the average value These kinetic parameters agreed within ex- of the amount of MPI& oxidized and the amount of MPH* perimental error by all three methods of de- and tyrosine formed after subtraction of the blank values.

472

LUNTE

AND KISSINGER TABLE

1

KINETICPARAMETERS Assay method Substrate Phe Phe MPH, MPH,

Kinetic parameter KM (mW V,, (nmol/min/mg KM (mM)* V,, (nmol/min/mg

protein)* protein)*

MPH4 0.52 7.8 0.039 11.2

* f * +

MPH2 0.05 0.4 0.04 0.5

0.56 8.7 0.032 9.9

+- 0.04 + 0.3 k 0.03 + 0.3

Tyrosine 0.57 8.6 0.029 9.2

k 0.03 f 0.3 -I 0.04 T!Z0.6

Average 0.55 8.4 0.033 10.0

+ f f f

0.05 0.4 0.04 0.6

’ The incubation contained 0.15 pmol of MPH,, and 2.2 mg of protein. * The incubation contained 1.0 pmol of phenylalanine and 2.6 mg of protein.

of MPH4 and phenylalanine initially present in the incubation mixture were varied and the ratio of MPH4 oxidized to MPH2 formed and the ratio of MPH4 oxidized to tyrosine formed were calculated to determine the stoichiometry of the reaction. At all initial concentrations of MPH4 and phenylalanine both ratios were found to be 1.0 within experimental error (Table 2). Since this is the stoichiometry previously reported (13), this indicates that determination of the loss of MPH4 or the formation of either product, MPH2 or tyrosine, is a valid method for determining the phenylalanine hydroxylase activity of a sample. Detection limits. Based on the oxidation of MPH4 or the formation of MPH*, rates up to 60 nmol/min can be accurately determined.

Also by these methods, as low a rate as 5 nmol/min can be determined. Using the formation of tyrosine, the same upper limit is achievable, however, much lower reaction rates can be determined due to the lower blank values. It is possible to determine as little as 0.1 nmol of tyrosine formed/min with the electrochemical detector. SUMMARY

The primary objective in the development of this methodology was to use the oxidation of the cofactor as a measure of enzyme activity. In this light, the use of LCEC offers advantages in selectivity and detectability over the previous spectroscopic method. When compared

TABLE 2 STOICHIOMETRYOFTHEPHENYLALANINEHYDROXYLASEREA~TION MPH4 (rmolh-4

Phe (wollml)

MPH., oxidized (nmol)

MPH* formed (nmol)

Tyrosine formed (nmol)

0.30 0.30 0.30 0.30 0.30 0.24 0.18 0.12 0.06

2.00 1.50 1.oo 0.50 0.26 2.00 2.00 2.00 2.00

2.44 2.15 1.94 1.54 0.9 1 2.25 1.70 1.39 0.76

2.59 2.34 1.96 1.47 0.99 2.23 1.78 1.36 0.80

2.44 2.27 2.04 1.46 0.96 2.23 1.67 1.39 0.80

Average

MPHJMPHz

MP&/Tyr

0.94 0.92 0.99 1.05 0.92 1.01 0.96 1.02 0.95

1.00 0.95 0.95 1.05 0.95 1.01 I .02 1.00 0.95

0.97

0.99

DETERMINATION

OF PHENYLALANINE

to the liquid chromatography/fluorescence methodology for the detection of tyrosine these parameters are essentially the same for the two procedures. However, LCEC requires relatively inexpensive equipment while allowing the simultaneous detection of tyrosine and the pterin cofactor. This LCEC methodology, with the ability to directly detect the oxidation of MPH4 as well as the formation of MPH2 and tyrosine, offers several other general advantages. The use of three measures of activity serves as an internal check of the assay system. No interferences can arise from the use of auxiliary enzymes as required in other methods. The method is also rapid and requires little sample. It has been shown that the response is linear over a wide range of activities and that the reaction rate is linear for several minutes. Since this methodology was demonstrated using unpurified liver samples these parameters should be improved by using more complete sample preparation procedures. Finally, the correct stoichiometry for the oxidation of MPH4 to MPH2 and the formation of tyrosine was ob-

HYDROXYLASE

ACTIVITY

413

tained indicating that all three methods of determining enzyme activity are analytically useful. REFERENCES 1. Kaufman, S. (1963) Proc. Nat. Acad. Sri. USA 50, 1085. 2. Kaufman, S. (1967) Advan. Enzymol. 35, 245. 3. Udenfiiend, S., and Cooper, J. R. ( 1952) J. Biol. Chem. 196, 227.

4. Waalkes, T. P., and Udenfriend, S. (1957) J. Lab. Clin. Med. 733. 5. Bailey, S. W., and Ayling, J. E. (1980) Anal. Biochem. 107, 156.

6. Guroff, G., Rhoads, C. A., and Abramowitz, A. (1967) Anal. Biochem. 21, 273. 7. Ayling, J., Pirson, R., Pirson, W., and Boehm, G. (1973) Anal. Biochem. 51, 80. 8. Kaufman, S. (1957) J. Biol. Chem. 226, 511. 9. Lunte, C. E., and Kissinger, P. T. (1983) Anal. Chem. 55, 1458. 10. Bailey, S. W., and Ayling, J. (1980) J. Biol. Chem. 255, 7774. 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 12. Nakata, H., and Fujisawa, H. (1980) Biochim. Biophys. Acfa 614, 313. 13. Storm, C. B., and Kaufman, S. (1968) Biochem. Biophys. Rex Commun. 38, 788.