Enzymatic Method for Determining Phenylalanine (Phenylalanine Assay)

Introduction

This assay protocol is suitable for the colorimetric/fluorometric detection of phenylalanine in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Phenylalanine Assay Kit (MAK005). Phenylalanine concentration is determined by a coupled enzyme assay, which results in the deamination of phenylalanine and the production of NADH which reacts with the probe resulting in a fluorometric (λex = 535/λem = 587 nm) product, proportional to the phenylalanine present. The linear range of detection for this assay is between 0.2–1.0 nmole.

Reagents

Phenylalanine Assay Buffer 25 mL
Catalog Number MAK005A

Tyrosinase 1 vl
Catalog Number MAK005B

Enzyme Mix 1 vl
Catalog Number MAK005C

Developer 1 vl
Catalog Number MAK005D

Phenylalanine Standard, 10 µmole 1 vl
Catalog Number MAK005E

Reagents and Equipment Required but Not Provided

96 well flat-bottom plate – it is recommended to use black plates with clear bottoms for fluorescence assays (Catalog Number M5686 or equivalent).

Fluorescence multiwell plate reader

10 kDa Molecular Weight Cut-Off (MWCO) Spin Filter (Catalog Number Z706345 or equivalent)

Preparation Instructions

Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Phenylalanine Assay Buffer – Allow buffer to come to room temperature before use.

Tyrosinase, Enzyme Mix, and Developer – Reconstitute each in 220 µL of Phenylalanine Assay Buffer. Mix well by pipetting, then aliquot and store at –20 °C. Keep cold while in use and protect from light. Use within 2 months of reconstitution.

Phenylalanine Standard – Reconstitute in 100 µL of water to generate a 10 mM (10 nmole/µL) Phenylalanine Standard Solution. Mix well by pipetting, then aliquot and store at –20 °C. Keep cold while in use.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure

All samples and standards should be run in duplicate.

Phenylalanine Standards for Fluorometric Detection
Dilute 10 µL of the 10 mM Phenylalanine Standard Solution with 990 µL of water to prepare a
0.1 mM (0.1 nmole/µL) standard solution. Add 0, 2, 4, 6, 8, 10 µL of the 0.1 mM phenylalanine standard solution into a 96 well plate, generating 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 nmole/well standards. Add Phenylalanine Assay Buffer to each well to bring the volume to 50 µL.

Sample Preparation
Serum samples should be deproteinized before use in the assay with a 10 kDa MWCO spin filter. 10–50 µL of deproteinized serum samples can be directly diluted to a final volume of 50 µL with the Phenylalanine Assay Buffer.

Tissue (20 mg) or cells (1 x 10⁶) can be homogenized in 100 µL of the Phenylalanine Assay Buffer. Centrifuge the samples at 13,000 x g for 10 minutes to remove insoluble material. Bring samples to a final volume of 50 µL with Phenylalanine Assay Buffer.

Notes: Samples other than serum may also be deproteinized with a 10 kDa MWCO spin filter prior to addition to the reaction.

For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

NADH and NADPH present in the samples may generate a background signal. To control for the background, a sample blank may be included for each sample by omitting the Enzyme Mix in the Reaction Mix.

Tyrosine present in the sample may generate a background signal. To control for tyrosine interference, the samples may be pretreated with 5 µL of Tyrosinase for 10 minutes at room temperature prior to start of the assay. Adjust the concentration of the Phenylalanine Assay Buffer in the reaction mix accordingly (see Table 1).

Assay Reaction

1. Set up the Reaction Mixes according to the scheme in Table 1. 50 µL of the appropriate Reaction Mix is required for each reaction (well).

2. Add 50 µL of the appropriate Reaction Mix to each of the blank, standard, and test wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 20 minutes at 37 °C. Protect the plate from light during the incubation.

3. Measure fluorescence intensity (λex = 535/λem = 587 nm).

Results

Calculations

The background for the assays is the value obtained for the 0 (blank) Phenylalanine standard. Correct for the background by subtracting the blank value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate phenylalanine standards to plot a standard curve.

Note: a new standard curve must be set up each time the assay is run.

Subtract the sample blank value from the sample reading to obtain the corrected measurement. Using the corrected measurement, the amount of phenylalanine present in the sample may be determined from the standard curve.

Concentration of Phenylalanine

Sa/Sv = C

Sa = Amount of phenylalanine in unknown sample (nmole) from standard curve
Sv = Sample volume (µL) added into the wells
C = Concentration of phenylalanine in sample

Phenylalanine molecular weight: 165.02 g/mole.

Sample Calculation

Amount of phenylalanine (Sa) = 5.84 nmole
Sample volume (Sv) = 50 µL

Concentration of phenylalanine in sample

5.84 nmole/50 µL = 0.1168 nmole/µL

0.1168 nmole/µL x 165.02 ng/nmole= 19.3 ng/µL

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.