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Tyrosine Decarboxylase - Assay

Source:
Streptococcus faecalis (NCTC6783)
CAS:
9002-09-9
EC:
4.1.1.25
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Tyrosine Decarboxylase

Method
Tyrosine Method

Tyrosine decarboxylase catalyzes the removal of the carboxyl group from tyrosine to produce tyramine and carbon dioxide, the tyramine produced is oxidized to p-hydroxybenzyl aldehyde and hydrogen peroxide by action of tyramine oxidase. The hydrogen peroxide reacts with 4- aminoantipyrine and TOOS in the presence of peroxidase to obtain quinone-imine dye which is measured at 570 nm2.

One unit results in the decomposition of one micromole of tyrosine per minute at 37°C under the specified conditions.

Reagents
  • McIlvain Buffer(phosphate-citrate), pH 6.0: Dissolve 1.79 gm sodium phosphate dibasic (MW 141.96) in 90ml reagent grade water. Add 775 mg citric acid (MW 210.14) and allow to dissolve. Adjust pH to 6.0 if necessary and bring to a final volume of 100 ml with water.
  • 10mM Potassium Phosphate Buffer pH 7.0: Dissolve 1.36 gm potassium phosphate monobasic (MW 136.09) in 900 ml reagent grade water. Adjust pH to 7.0 and bring to a final volume of 1.0LT.
  • 1mM 4-aminoantipyrine: Dissolve 8.1 mg 4-AAP (MW 203.2) in 40 ml reagent grade water.
  • 10mM TOOS: Dissolve 33.1mg N-Ethyl-N-(2-hydroxy-3-sulfopropyl) m-toluidine (TOOS) in 10 ml reagent grade water. Handle with caution, consult MSDS. Use Dojindo Labs Japan (OC13-10 ) or equivalent.
  • 40 u/ml Peroxidase (Toyobo PEO 301 or equivalent): Dilute as needed to prepare a 40u/ml solution in cold 10mM phosphate buffer pH 7.0.
  • 20 u/ml Tyramine Oxidase (Toyobo TYO-301 or equivalent): Dilute as needed to prepare a 20 u/ml solution in cold 10mM phosphate buffer pH 7.0
  • 0.2mM Pyridoxal-5-phosphate buffer: Dissolve 4.9mg pyridoxal phosphate (MW 247.1) in 100ml 0.01N HCl.
  • 10mM Tyrosine: Dissolve 18.1 mg tyrosine (MW 181.2) in 0.5ml 1N HCl and bring to a final volume of 10ml with reagent grade water.
  • Enzyme Dilution Buffer: 10mM potassium phosphate pH 7.0 with 5mM EDTA. Dissolve 558mg EDTA (MW 372.25) in 250 ml 10mM potassium phosphate buffer. Adjust pH to 7.0 if necessary and bring to a final volume of 300 ml with 10mM potassium phosphate buffer.
Enzyme

Suspend at 1 mg/ml in Enzyme Dilution Buffer. Dilute as needed to obtain rates near 0.080 ΔA/min. Run one rate at a time for at least 2 minutes.

Procedure

Set up spectrophotometer for 37°C and 570 nm.

Reaction mixture is: 

  • McIlvain Buffer:  0.9ml
  • 1mM 4AAP:  0.9ml
  • 10mM TOOS:  0.3ml 
  • 40 u/ml peroxidase: 0.15ml 
  • 20 u/ml TYO: 0.15ml 
  • 0.2mM pyridoxal-5-phosphate: 0.15ml 
  • 10mM tyrosine: 0.3ml

Equilibrate reaction mixture in cuvettes at 37°C prior to adding sample (0.1ml or 0.15ml sample volume).

 

Calculation

millimolar extinction coefficient of TOOS=32
amount of dye from one H2O2 molecule = 1⁄2 molecule

Tyrosine Decarboxylase Products

Description
Activity
Code
Cat. #
Size
Price
Description
Tyrosine Decarboxylase
Source:
Streptococcus faecalis (NCTC6783)
HOLOenzyme. Dried cells.
Store at -20°C.
…
Dry Ice required
Activity
≥0.2 units per mg dry weight
Code
TYD
LS004966
25 un
$55.00
LS004964
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 500 x 1 un. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
Description
Tyrosine Decarboxylase, Apoenzyme
Source:
Streptococcus faecalis (NCTC6783)
APOenzyme. Dried cells grown in B6 deficient media.
Store at -20°C.
…
Dry Ice required
Activity
Activates to at least 0.2 units per mg
Code
TYDAPO
LS004968
250 mg
$68.00
LS004970
1 gm
$216.00
LS004973
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 20000 x 1 mg. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.