CODE No.:B2P111

PHOSPHOFRUCTOKINASE (PFK)

[EC 2.7.1.11]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 100 U/mg protein
Contaminants	:	(as PFK activity = 100%)
		Adenylate kinase	<0.01%
		ATPase	<0.01%
		6-Phosphogluconate dehydrogenase	<0.01%
		Glutathione reductase	<0.01%
		Phosphoglucomutase	<0.01%
		Glucose phosphate isomerase	<0.01%

PROPERTIES

Molecular weight	:	ca. 74,000
Subunit molecular weight	:	ca. 34,000
Optimum pH	:	9.0	(Fig.1)
pH stability	:	6.5 - 10.0	(Fig.2)
Isoelectric point	:	6.0 - 6.2
Thermal stability	:	No detectable decrease in activity up to 50°C.	(Fig.3, 4)
Michaelis constants	:	(91mM Tris-HCl buffer, pH 9.0, at 30°C)
		Fructose 6-phosphate	1.6mM
		ATP	0.035mM
Activator	:	K+, (NH4)2SO4
Inhibitors	:	PEP, Citrate

STORAGE

stable at -20°C for at least one year

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reactions.

Unit Definition

One unit of activity is defined as the amount of PFK that forms 1 μmol of fructose 1, 6-bisphosphate per minute at 30°C.

Solutions

1. Buffer solution ; 100mM Tris-HCl, pH 9.0
2. ATP solution ; 100mM (0.605 g ATP disodium salt·3H₂O/(8.2 mL distilled water + 1.8 mL 1 N-NaOH))
3. Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10mL distilled water)
4. NADH solution ; 13.1 mM (0.100 g NADH disodium salt·3H₂O/10mL distilled water)
5. Fructose 6-phosphate (F6P) solution ; 500mM (1.55 g F6P disodium salt/10mL distilled water)
6. KCl solution ; 2.5 M (16.64g KCl/100mL distilled water)
7. MgSO₄ solution ; 100mM (2.47g MgSO₄·7H₂O/100mL distilled water)
8. Pyruvate kinase (PK) ; (from rabbit muscle, Roche Diagnostics K.K., No. 128 155) crystalline suspension in 3.2 M (NH₄)₂SO₄ solution (10 mg/mL) approx. 200 U/mg at 25°C
9. Lactate dehydrogenase (LDH) ; (from hog muscle, Roche Diagnostics K.K., No. 127 221) 50% glycerol solution (2.5 mg/2.5 mL) approx. 550 U/mg at 25°C

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50mM potassium phosphate buffer, pH 8.0.

Procedure

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

   Solution I	27.33mL	Solution VI	0.06mL
   Solution II	0.30mL	Solution VII	0.60mL
   Solution III	0.39mL	Solution VIII	0.06mL
   Solution IV	0.60mL	Solution IX	0.06mL
   Solution V	0.60mL

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 340 nm per minute (ΔAbs₃₄₀) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH (cm²/μmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Hengartner, H., and Harris, J.I.; FEBS Lett., 55, 282 (1975)

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