PROTOCATECHUATE 3,4-DIOXYGENASE from Pseudomonas sp.

PCO-302

Protocatechuate:oxygen 3,4-oxidoreductase (decyclizing) (EC 1.13.11.3)
Protocatechuate + O₂ ► ß-Carboxymuconate

PREPARATION and SPECIFICATION
Appearance:	Light brown amorphous powder, lyophilized
Activity:	GradeⅢ 3.0U/mg-solid or more
Contaminant:	NADPH oxidase ≤1.0×10⁻¹%
Stabilizers:	Sugars

PROPERTIES
Stability:	Stable at -20°C (Fig.1)
Molecular weight :	approx. 600,000 (by gel filtration)
Michaelis constant:	1.85×10⁻⁵M (Protocatechuate)
Structure:	Protein with nonheme iron
Inhibitors:	Ag⁺, Hg⁺⁺
Optimum pH :	9.0(Fig.2)
Optimum temperature :	60-65°C(Fig.3)
pH Stability :	pH 6.0-9.5 (25°C, 72hr)(Fig.4)
Thermal stability :	below 55°C (pH 7.5, 1hr)(Fig.5)
Effect of various chemicals :	(Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of choline esterase when coupled with p-hydroxybenzoate hydroxylase (HBH-311).

ASSAY

Principle:

protocatechuate 3,4-dioxygenase

Protocatechuate+O₂ ► ß-Carboxymuconate

The disappearance of protocatechuate is measured at 290nm by spectrophotometry.

Unit definition:

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

Method:

Reagents
A. Tris-acetate buffer, pH 7.5:	50mM[Dissolve 6.1g of Tris (MW=121.14) in ca.800ml of H₂O and, after adjusting pH to 7.5 at 25°C with 0.2M acetic acid, fill up to 1,000ml with H₂O.]
B. Protocatechuate acid solution:	0.4mM[Dissolve 6.16mg of protocatechuate in ca.80ml of buffer (A) and, after adjusting pH to 7.5 at 25°C with 1.0N KOH, fill up to 100ml with buffer (A).] (Should be prepared fresh)

Procedure

Concentration in assay mixture
Tris-acetate buffer	50 mM
Protocatechuate	0.39 mM

1. Pipette 3.0ml of protocatechuate solution (B) into a cuvette (d=1.0cm) and equilibrate at 37°C for about 5 minutes.

2. Add 0.05ml of the enzyme solution* and mix by gentle inversion.

3. Record the decrease in optical density at 290nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37°C, and calculate the ΔOD per minute from the initial linear 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 (A) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold diluent (A) (1.0mg/ml or more) and dilute to 0.2-0.8U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test-ΔOD blank)×Vt×df

Volume activity (U/ml) = = ΔOD/min×16.1×df

3.8×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt: : Total volume (3.05ml)
Vs: : Sample volume (0.05ml)
3.8: : Millimolar extinction coefficient of protocatechuate (㎠/micromole)
1.0: : Light path length (cm)
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

H.Fujisawa and O.Hayashi; J.Biol.Chem., 243, 2673 (1968)

**Table 1. Effect of Various Chemicals on Protocatechuate 3,4-oxidoreductase**
[The enzyme dissolved in 50mM Tris-Acetate buffer (5U/ml) was incubated with each chemical at 30°C for 1hr.]
Chemical	Concn.(mM)	Residual activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	NaF	1.0	100
Metal salt	1.0		NaN₃	1.0	98
AgNO₃		26	EDTA	5.0	98
BaCl₂		97	Borate	50	97
CaCl₂		97	SDS	0.05%	101
CoCl₂		97	Brij 35	0.10%	103
CuSO₄		95	Tween 20	0.10%	100
FeSO₄		79	Na-cholate	0.10%	101
MgSO₄		100
MnCl₂		100
NiCl₂		100
ZnCl₂		95

EDTA, ethylenediaminetetraacetate; SDS, sodium dodecyl sulfate.

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