PHOSPHOENOLPYRUVATE CARBOXYLASE from Microorganism

PPC-301

PREPARATION and SPECIFICATION
Appearance:	White amorphous powder, lyophilized
Activity:	GradeⅢ 5.0U/mg-solid or more
Contaminants:	Lactate dehydrogenase ≤1.0×10⁻³% Pyruvate kinase ≤0.5%
Stabilizers:	BSA, sugar alcohols

PROPERTIES
Stability:	Stable at -20°C for at least One year (Fig.1)
Molecular weight :	approx. 390,000 (by gel filtration)
Isoelectric point:	6.0±0.1
Structure:	4 Subunits (M.W.100,000) per enzyme molecule
Michaelis constant:	1.9×10⁻⁴M (Phosphoenolpyruvate)
Optimum pH :	7.5-8.0(Fig.2)
Optimum temperature :	60°C(Fig.3)
pH Stability :	pH 5.0-8.0 (25°C, 24hr)(Fig.4)
Thermal stability :	below 40°C (pH 7.0, 15min)(Fig.5)

APPLICATIONS

This enzyme is useful for enzymatic determination of carbon dioxide when coupled with malate dehydrogenase (MAD-201, MAD-211) in clinical analysis.

ASSAY

Principle:

phosphoenolpyruvate carboxylase

Phosphoenolpyruvate+CO₂+H₂O ► Oxaloacetate+Pi

malate dehydrogenase

Oxaloacetate+NADH+H⁺ ► L-Malate+NAD⁺
The disappearance of NADH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the oxidation of one micromole of NADH per minute under the conditions described below.

Method:

Reagents
A. Buffer solution:	0.1M Tris-HCl Buffer, pH 8.0
B. Na₂CO₃ solution:	0.1M[Dissolve 1.06g of Na₂CO₃(MW=105.99) /100ml of H₂O]
C. K-Phosphoenolpyruvate: solution	32mM [Dissolve 33.0mg of PEP・K (MW=206.1) /5ml of H₂O](Should be prepared fresh)
D. MgSO₄ solution:	1M[Dissolve 4.93g of MgSO₄・7H₂O(MW=246.48)/20 ml of H₂O]
E. NADH solution:	1.4mM[Dissolve 5.34mg of NADH・3H₂O(MW=763)/5ml of H₂O]
F. MDH solution:	ca.100U/m[l Dissolve malate dehydrogenase (TOYOBO GradeII) to approx.100U/ml with 20mM Tris-HCl Buffer,pH 8.0](Should be prepared fresh)
G. Enzyme diluent:	20mM K-phosphate buffer, pH 7.0

Procedure

Concentration in assay mixture
K-Phosphoenolpyruvate	3.1 mM
Tris-HCl	57 mM
Na₂CO₃	9.7 mM
MgSO₄	9.7 mM
NADH	0.14mM
MDH	ca.9.7 U/ml
K-Phosphate	0.65mM

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 30°C for about 5 minutes.

1.77ml Buffer solution (A)
0.3 ml Na₂CO₃ solution (B)
0.3 ml K-Phosphoenolpyruvate solution (C)
0.03ml MgSO₄ solution (D)
0.3 ml NADH solution (E)
0.3 ml MDH solution (F)

2. Add 0.1ml of the enzyme solution* and mix by gentle inversion.

3. Record the decrease in optical density at 340nm against water for 3 to 4 minutes in a spectro-photometer thermostated at 30°C, and calculate the ΔOD per minute from initial liner portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent (G) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (G) and dilute to 0.2-0.7U/ml with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min(ΔOD test-ΔOD blank)×Vt×df

Volume activity (U/ml) = = ΔOD/min×4.98×df

6.22×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt: : Total volume (3.1ml)
Vs: : Sample volume (0.1ml)
6.22: : Millimolar extinction coefficient of NADH (㎠/micromole)
1.0: : Light path length (cm)
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

W.Wilson, P.Jesyk, R.Rand and R.D.Bevill; Clin.Chem.,19, 640(1973)
R.L.Forrester, L.J.Wataji, D.A.Silverman and K.J.Pierre; Clin.Chem.,22, 243(1976)

**Table 1. Effect of Various Chemicals on Phosphoenolpyruvate carboxylase**
[The enzyme solution dissolved in 20mM K-phosphate buffer, pH 7.0 (20U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical	Concn.(mM)	Residual activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	PCMB	0.1	80
Metal salt	2.0		NEM	2.0	87
MgCl₂		105	IAA	2.0	90
CaCl₂		105	Hydroxylamine	2.0	95
Ba(OAc)₂		103	EDTA	5.0	100
FeCl₃		92	o-Phenanthroline	2.0	103
CoCl₂		106	α,α′-Dipyridyl	2.0	109
MnCl₂		107	Borate	5.0	103
ZnSO₄		103	NaF	2.0	106
Cd(OAc)₂		104	NaN₃	2.0	106
NiCl₂		0	Triton X-100	0.10%	111
CuSO₄		0	Brij 35	0.10%	110
Pb(OAc)₂		105	Tween 20	0.10%	112
AgNO₃		0	Span 20	0.10%	109
HgCl₂		0	Na-cholate	0.10%	108
MIA	2.0	60	SDS	0.05%	1
2-Mercaptoethanol	2.0	101	DAC	0.05%	99

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate;
IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylallkylammonium chloride.

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