Team:Imperial College London/Wetlab/Protocols/PAH

=PAH=

Introduction
This assay allows us to quantify the amount of tyrosine in the sample, thanks to spectrophotometry. The enzyme of interest, PAH, degrades Phenylalanine into Tyrosine. By shining light at the optimal wavelength of the enzyme cofactor, we obtain absorbance readings. Using the Beer-Lambert law, we can then determine the concentration of the amino acid. Subsequently plotting this information (absorbance readings) against time, allows us to determine the activity rate of the enzyme. However, we need to assume that only the PAH enzyme is able to catalyse this reaction.

Considerations
In order to carry this assay out correctly, we need to lyse the cells to obtain what is called a crude extract. This will be a 'soup' of all proteins -including the PAH enzyme- that were contained in the bacteria. Selectiveness for Tyrosine is introduced by choosing the wavelength of the beam to be the optimal wavelength of the enzyme cofactor. This step has been added to the protocol below in red.

Abbreviations
6-MTHP = 6-Methyltetrahydropterine

6-MDHP = 6-Methyldihydropterine

PAH = L-Phenylalanine Hydroxylase

CONDITIONS
T = 25°C, pH = 7.2, A450nm, Light Path = 1 cm

METHOD
Colorimetric: Spectrophotometry @ 450nm

REAGENTS
A) 200 mM Tris HC1 Buffer, pH 7.2 at 25EC

Prepare 100 ml in deionized water using Trizma Base, Sigma Prod. No. T-1503. Adjust to pH 7.2 at 25°C with 1 M HCl.

B) 4.0 mM L-Phenylalanine Solution (PHE)

Prepare 30 ml in Reagent A using L-Phenylalanine, Sigma Prod. No. P-2126.

C) Catalase Enzyme Solution (CAT)

Prepare 20 ml of a solution containing 4000 units/ml of Catalase, Sigma Stock No. C-100, in cold Reagent B (PHE). PREPARE FRESH.

D) 16.65 mM DL-Dithiothreitol Solution (DTT)

Prepare 25 ml in deionized water using DL-Dithiothreitol, Sigma Prod. No. D-0632.

E) 1.33 mM 6-Methyltetrahydropterine Solution (6-MTHP)

Prepare 10 ml in Reagent D (DTT) using DL-6-Methyl-5,6,7,8-Tetrahydropterine Dihydrochloride, Sigma Prod. No. M-4758. PREPARE FRESH.

F) 5% (v/v) Trichloroacetic Acid Solution (TCA)

Prepare 25 ml in deionized water using Trichloroacetic Acid, 6.1 N Solution, Sigma Stock No. 490-10.

G) 20% (v/v) Nitric Acid Solution with 0.05% (w/v) Sodium Nitrite (Nitric Acid)

Prepare 20 ml in deionized water using Nitric Acid, Aldrich Stock No. 25811-3 and Sodium Nitrite, Sigma Prod. No. S-2252. Dissolve the Sodium Nitrite in deionized water before adding to the Nitric Acid solution.

H) 0.1% (v/v) Nitrosonaphthol Solution (NNS)

Prepare 20 ml in Reagent I (NaOH) using 1-Nitroso-2-Naphthol, Sigma Prod. No. N-3765.1

I) 100 mM Sodium Hydroxide Solution (NaOH)

Prepare 50 ml in deionized water using Sodium Hydroxide Solution, 1.0 N, Sigma Stock No. 930-65.

J) 5.0 mM L-Tyrosine Standard Solution (TYR Std)

Prepare 10 ml in deionized water using L-Tyrosine Free Base, Sigma Prod. No. T-3754.

K) Phenylalanine Hydroxylase Enzyme Solution

Immediately before use, prepare a solution containing 0.2 - 1.0 unit/ml of Phenylalanine Hydroxylase in cold Reagent A.

PROCEDURE
0) Sonicate your cells on ice at ~20KHz on 2 seconds on/off alternating cycles (2 seconds on, 2 seconds off etc) for 7 minutes

1) Prepare 6 x 2mL Eppendorf tubes.

2) Pipette (in microliters) the following reagents into suitable containers:

3) Mix by swirling and equilibrate to 25°C. Then add:

4) Immediately mix by swirling, and incubate at 25°C for exactly 8 minutes. Then add:

5) Mix by swirling and heat at 55°C for 30 minutes. Cool to 25°C and centrifuge for 3 minutes. Transfer the solutions to suitable cuvettes and record the A450nm for the Test, Blank, Standards and Standard Blank with a suitable spectrophotometer.

Standard Curve
DA450nm Standard = A450nm Standard - A450nm Standard Blank

Prepare a standard curve by plotting the DA450nm for the Standards versus micromoles of Tyrosine.

Sample Determination
DA450nm Sample = A450nm Test - A450nm Blank

To determine the total micromoles of L-Tyrosine produced using the Standard curve.

Units/ml enzyme= ((mmoles L-Tyrosine produced)(df))÷((0.02) (8))

df = Dilution factor

8 = Time correction factor (in minutes) as per Unit Definition

0.02 = Volume (in milliliters) of enzyme used in the assay

Units/mg solid = units/ml enzyme ÷  mg solid/ml enzyme

Units/mg protein = units/ml enzyme ÷ mg protein/ml enzyme

UNIT DEFINITION
One unit will convert 1.0 mmole of L-phenylalanine to L-tyrosine per min at pH 7.2 and 25°C, using DL-methyl-5,6,7,8-tetrahydropterine as cofactor.

FINAL ASSAY CONCENTRATION
In a 1.00 ml reaction mix, the final concentration are 104 mM Tris, 2 mM L-phenylalanine, 2000 units catalase, 5.0 mM DL-dithiothreitol, 0.40 mM DL-6-methyl-5,6,7,8-tetrahydropterine and 0.004 - 0.02 unit L-phenylalanine hydroxylase.