Tyrosine Decarboxylase Assay

Assay for TYD Enzyme:

Method: The assay used here is based on that of Gunsalus and Smith (Methods in Enzymology, III, (Colowick, S., and Kaplan, N., eds.), Academic Press, NY, 963, 1957). One unit yields one micromole of CO₂ per minute from L-tyrosine at 37°C and pH 5.5 under the specified conditions. Reagents

0.8 M Sodium acetate, pH 5.5
0.074 M Sodium acetate, pH 5.5
0.0065 M L-tyrosine in reagent grade water. Dissolve by warming to 80°C while stirring. Equilibrate to 37°C. (If solution cools crystals may form).

Enzyme

Dissolve enzyme in 0.074 M sodium acetate, pH 5.5 at a concentration of 2 mg/ml. Mix to uniformity immediately before withdrawing aliquots.

Procedure

Into the main well of the Warburg flasks pipette the following:

0.8 M Sodium acetate, pH 5.5 0.2 ml

0.0065 M L-tyrosine at 37°C 2.3 ml

Pipette 0.5 ml of the enzyme suspension into the side arm of the flasks. Include one flask containing no enzyme as a blank and a flask containing no enzyme to serve as a blank and a flask containing 3.0 ml of reagent grade water to serve as a thermal barometer. Attach flasks to manometer. After 10 minutes of equilibration, close the manometers, tip in and mix the substrate and replace the flasks in the bath.

Determine the micromoles of CO₂ released at approximately 5 minute intervals for 30 minutes. Plot micromoles CO₂ released versus reaction time and determine micromoles released per minute from the linear portion of the curve

Calculation

Assay for TYD-APO:

0.8 M Sodium acetate, pH 5.5: Add 11.45ml glacial acetic acid (MW 60.05, specific gravity 1.05) to 200ml reagent grade water. Adjust pH to 5.5 with 5N NaOH and bring to a final voulme of 250ml with reagent grade water.
0.222 M Sodium acetate, pH 5.5: Dilute 10ml of 0.8 M Sodium acetate, pH 5.5, with 26ml of reagent grade water.
0.0065 M L-tyrosine in reagent grade water. Dissolve by warming to 80°C while stirring. Equilibrate to 37°C. (If solution cools, crystals may form. Reheat to dissolve.)
Pyridoxal Phosphate(B-6) Standard, freshly prepared.
B-6 Solution #1: Dissolve 5mg in 100ml reagent grade water, to obtain a concentration of 50 µg/ml
B-6 Solution #2: Dilute B-6 Solution #1 further 500X immediately before use to obtain a concentration of 100ng/ml.

Enzyme

Suspend at 5 mg/ml in reagent grade water. Mix to uniformity before withdrawing aliquots.

Procedure

Set up pyridoxal phosphate curve in test tubes to give 0, 5 ng, 10 ng, 15 ng and 5 ug curve in Warburg flasks. Each Warburg flask will contain 1 mg of enzyme.

Tube Freshly
mixed
5 mg/ml
enzyme 0.222 M
sodium
acetate reagent
grade
water pyridoxal
phosphate
standard
B-6 #2 pyridoxal
phosphate
standard
B-6 #2

0 0.4 ml 0.3 ml 0.3 ml ----- -----

5 ng 0.4 ml 0.3 ml 0.2 ml 0.1 ml -----

10 ng 0.4 ml 0.3 ml 0.1 ml 0.2 ml -----

15 ng 0.4 ml 0.3 ml ----- 0.3 ml -----

5 ug 0.4 ml 0.3 ml 0.1 ml ----- 0.2 ml

Mix well. Transfer 0.5 ml from each tube to the side arm of a corresponding Warburg flask. Into each main well of the Warburg flasks pipette the following:

0.8 M Sodium acetate, pH 5.5 0.2 ml

0.0065 M L-tyrosine at 37¡C 2.3 ml

Include one flask containing no enzyme as a blank and a flask containing 3.0 ml of reagent grade water to serve as a thermal barometer. Attach flasks to manometer. After 10 minutes of equilibration close the manometers. Re-equilibrate 10 minutes with manometers closed, and read to establish zero time point. Tip in and mix the substrate and replace the flasks in the bath. Determine the micromoles of CO₂ released at approximately 5 minute intervals for 30 minutes. Plot micromoles CO₂ released versus reaction volume for each tube 0 - 15 ng and unknown samples.
Spec:

Activates to * 0.1 units/mg dw with 5µgrams of pyridoxal phosphate
Demonstrated done response.
Gives little or no background.

Form: Dried cells

Calculation

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