Phosphoenolpyruvate Carboxylase

Product description

This enzyme is useful for enzymatic determination of CO₂ when coupled with MDH (HH1802) in clinical analysis.

Product specifications

Appearance	White amorphous powder，lyophilized
Stabilizers	BSA, sugar alcohols
Powder activity	≥5.0U/mg
Pyruvate kinase	≤0.05%
Lactate dehydrogenase	≤0.001%

Properties

Source	Microorganism
EC number	EC 4.1.1.31
Molecular weight	104.4kD （SDS-PAGE）
Isoelectric point	pI(6.0±0.1)
Optimum pH	pH5.0-9.0	Fig. 1
Optimum temperature	60℃	Fig. 2
pH stability	pH 5.5-9.0 (25℃, 24hr)	Fig. 3
Thermal stability	Below 45℃(pH7.0, 30min)	Fig. 4
Storage	-25～-15℃

Fig. 1 Optimum pH

50mM buffer solution: pH 4.0-5.5, NaAc-HAc;
pH 6.0-8.0, PB-K; pH 7.5-9.0, Tris-HCl.

Fig. 2 Optimum temperature

Reaction in 20mM PB-K pH 7.0.

Fig. 3 pH Stability

25℃, 24 h-treatment with 20mM buffer solution: pH 4.0-5.5, NaAc-HAc;
pH 5.0-8.0, PB-K; pH 8.0-9.0, Tris-HCl.
Protein concentration: 1mg/mL

Fig. 4 Thermal stability

30min-treatment with 20mM K-phosphate pH 7.0.
Protein concentration: 1mg/mL

Activity assay method

Principle

The consumption of NADH is measured at 340 nm by spectrophotometry.

Unit definition

1U is defined as the the amount of enzyme which consumes 1μmol of NADH per min under the conditions described below..

Reagents

1. Reagent A: 100mM Tris-HCl (pH8.0)

2. Reagent B (100mM Na₂CO₃): Dissolve 1.06g of Na₂CO₃ (MW＝105.99) in 100ml of pure water.

3. Reagent C (32mM K-Phosphoenolpyruvate): Dissolve 33.0mg of PEP・K (MW＝206.1) in 5ml of pure water, should be prepared fresh.

4. Reagent D (1M MgSO₄): Dissolve 4.93g of MgSO₄・7H₂O (MW＝246.48) in 20 ml of pure water.

5. Reagent E(1.4mM NADH): Dissolve 10mg of NADH (MW＝709.4, HH3201) in 10ml of pure water.

6. Reagent F (100U/mL MDH): Dissolve MDH (HH1802) to approx.100U/ml with 20mM Tris-HCl Buffer (pH 8.0), should be prepared fresh.

7. Reaction I: 1. Prepare the following reaction mixture in a 50ml tube, mix well, and set aside for use in ice bath, , should be prepared fresh.

Reagent	Volume	Final concentration
A	17.7mL	59mM Tris-HCl pH8.0
B	3.0mL	10mM Na₂CO₃
C	3.0mL	3.2mM PEP・K
D	0.3mL	10M MgSO₄
E	3.0mL	0.14mM NADH
F	3.0mL	10U/mL MDH

8. Reagent II: Enzyme diluent, 20mM PB-K pH7.0, store at 2-8℃.

9. Dissolution: Reconstitution the powder with water to 50mg/ml, and set aside for use in ice bath.

10. Sample: The enzyme was diluted to 0.2-0.7 U/mL with the Reagent II, and store on ice.

Procedure

1. Add 900μL Reagent I to the 1 mL cuvette and preheat it at 30℃ for 5 min;

2. Add 30μL Enzyme solution to the cuvette and mix;

3. Record the ΔA_s/min at 340 nm from 3rd to 4th minute in a spectrophotometer thermostated at 30℃.

4. At the same time, measure the blank rate ΔA_b/min by using the same method as the test except that the Reagent II is added instead of the enzyme solution.

∆A/min =∆A_s/min - ∆A_b/min

Calculation

Vt: Total volume 0.93mL

Vs: Sample volume 0.03mL

d : Light path length 1cm

df: Dilution factor

C: Powder concentration in dissolution (mg/ml)

6.22: Millimolar extinction coefficient of NADH under 340nm (cm²/μmol)