CODE No.:B2P311

PHOSPHOTRANSACETYLASE (PTA)

[EC 2.3.1.8]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 5,000 U/mg protein
Contaminants	:	(as PTA activity = 100%)
		Acetate kinase	<0.01%
		Adenylate kinase	<0.01%
		Lactate dehydrogenase	<0.01%

PROPERTIES

Molecular weight	:	ca. 70,000
Subunit molecular weight	:	ca. 35,000
Optimum pH	:	7.5	(Fig.1)
pH stability	:	7.0 - 11.0	(Fig.2)
Isoelectric point	:	4.5
Thermal stability	:	No detectable decrease in activity up to 50°C.	(Fig.3, 4)
Michaelis constants	:	(87mM Tris-HCl buffer, pH 7.5, at 30°C)
		Coenzyme A	0.4mM
		Acetyl Phosphate	1.1mM

STORAGE

stable at -20°C for at least one year

APPLICATION

The enzyme is useful for determination of CoA or acetate.

ASSAY

Principle

The change in absorbance is measured at 233 nm according to the following reaction.

Unit Definition

One unit of activity is defined as the amount of PTA that forms 1 µmol of acetyl-CoA per minute at 30°C.

Solutions

1. Buffer solution ; 100mM Tris-HCl, pH 7.5
2. CoA solution ; 6.4 mM (50 mg CoA trilithium salt/10mL distilled water)
3. Acetylphosphate solution ; 217 mM (0.400 g acetylphosphate potassium lithium salt/10mL distilled water)
4. Ammonium sulfate (AmS) solution ; 1 M (13.2 g AmS/100mL distilled water)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 20 U/mL with 50mM Tris-HCl buffer, pH 8.0.

Procedure

1. Prepare the following reaction mixture and pipette 3.00mL of reaction mixture into a cuvette.

   Solution I	26.0mL	Solution III	1.0mL
   Solution II	2.0mL	Solution IV	1.0mL

2. Incubate at 30°C for about 3 minutes.
3. Add 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 233 nm per minute (ΔAbs₂₃₃) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 4.44 ; differential millimolar extinction coefficient between acety-CoA and CoA (cm²/µmol)
• *Protein concentration ; determined by Bradford's method

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