(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-209REACH適合品

OXALOACETATE DECARBOXYLASE [OACⅡ]

from Pseudomonas sp.
(Oxaloacetate carboxy–lyase, EC 4.1.1.3)
(Long–chain–fatty–acid–CoA ligase)

Oxaloacetate → Pyruvate + CO₂

Preparation and Specification

Appearance: : White to off white amorphous powder, lyophilized

Specific activity: : More than 2300 U/mg solid

Contaminants: :

AST (GOT): Less than 0.005 % (U/U)

Catalase: Less than 0.3 % (U/U)

Properties

Molecular weight: : 31 kDa (SDS–PAGE) , 120 kDa (Superdex 200)

Isoelectric point: : pH 5.16

Michaelis constants: : Oxaloacetate 3.3 × 10^-3M

Optimum pH: : 7.5–8.5Figure 1

pH stability: : 7.5–9.0 (50℃, 3 hr.) Figure 2

Optimum temperature: : 40–50℃Figure3

Thermal stability: : Stable at 50℃ and below (pH 7.0, 10 min) Figure4

Storage stability: : At least one year at －20℃

Activators: : Mg²⁺, Mn²⁺

Inhibitors: : Sodium dodecylsulfate, Sodium laurylbenzen sulfonate

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of AST when coupled with pyruvate oxidase (T–45)

	AST
Aspartic acid + α － ketoglutarate	→	Oxaloacetate + Glutamic acid
	OACⅡ
Oxaloacetate	→	Pyruvate + CO₂
	POPG
Pyruvate + O₂ + H₂O	→	Acetylphosphate + CO₂ + H₂O₂
	POD
2 H₂O₂ + 4－AA + Phenol	→	Quinoneimine dye + 4 H₂O

Fig.1 pH Optimum

●: 3,3-Dimethylglutarate-NaOH buffer
〇: Tris-HCI buffer
△: Glycine-NaOH buffer

Fig.2 pH Stability

50℃, 3 hr.
●: 3,3-Dimethylglutarate-NaOH buffer
〇: Tris-HCI buffer
△: Glycine-NaOH buffer

Fig.3 Optimum Temperature

pH 8.0
0.1 M TEA-NaOH buffer

Fig.4 Thermal Stability

pH 7.0, 10 min.
50 mM phosphate buffer

Assay

Principle

The assay is based on the decrease in absorbance at 340 nm as the consumption of NADH proceeds in the following reactions:

	OAC Ⅱ
Oxaloacetate	→	Pyruvate+CO₂
	LDH
Pyruvate+NADH+H⁺	→	Lactate+NAD⁺

LDH: Lactate dehydrogenase
NADH: Nicotineamide adenine dinucleotide

Unit definition

One unit is defined as the amount of enzyme which produces 1 μmole of pyruvate per minute at 25℃ under the conditions specified in the assay procedure.

Reagents

Reaction mixture

0.1 M TEA–NaOH buffer pH 8.0 0.90 ml

10 mM MnCl2 solution 0.10 ml

6.5 mM NADH solution 0.10 ml

Distilled water 0.90 ml
550 U/ml LDH solution
Mix 0.1 ml of LDH solution (10 mg/2 ml, 2,750 U/ml) with 0.4 ml of 10 mM Tris–HCl buffer pH 7.0
Substrate solution (50 mM oxaloacetate solution)
Dissolve 33 mg of oxaloacetate with 5 ml of 0.2 M Tris–HCl buffer pH 9.0.
Enzyme dilution buffer
10 mM Tris–HCl buffer pH 8.0
Reagents
TEA (Triethanolamine, HCl salt) : Merck Co.
MnCl₂ ・ 4H₂O:

FUJIFILM Wako Pure Chemical Corporation
Special grade #133–00725

NADH (2Na・3H₂O, Reduced form) :
Kyowa Hakko Co., Ltd.
LDH: Roche Diagnostics GmbH #10 127 876 001
Oxaloacetate:
FUJIFILM Wako Pure Chemical Corporation #150-00411

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration to within 0.5–1.4 U/ml.

Procedure

Pipette accurately 2.0 ml of reaction mixture, 20 μl of LDH solution and 50 μl of enzyme solution into a samll test tube and preincubate at 25℃.

※ In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution.
After 5 min, add 100 μl of substrate solution and mix to start the reaction at 25℃.
After starting the reaction, measure the rate of decrease per minute in absorbance at 340 nm. The rate must be measured within the linear portion of the absorbance curve.
Absorbance sample : As/min

blank : Ab/min

△A/min = (As/min－Ab/min) ≦ 0.15 Abs/min

Calculation

Activity (U/mg of powder) = {(△ A/min)/(6.22)} × 2.17/0.05 × 1/x

6.22 :	millimolar extinction coefficient of NADH at 340 nm (cm²/ μmole)
2.17 :	final volume (ml)
0.05 :	volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution ( mg/ml)

Storage

Storage at -20℃ in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition.

References

Horton, A. A. and Kornberg, H. L. (1964) Biochem.
Biophys. Acta, 89, 381–383.
Schmitt, A., Bottke, I. and Siebert, G. (1966) Hoppe–Seyler’s Z. Physiol. Chem., 347, 18–34.

OAC Ⅱ活性測定法 (Japanese)

試薬液

反応試薬混合液

0.1M TEA–NaOH 緩衝液 pH8.0
0.90 ml

10mM 塩化マンガン溶液
0.10 ml

6.5mM NADH 溶液
0.10 ml

精製水 0.90 ml
550U/ml LDH 溶液
ロシュ製LDH 溶液 (10mg/2ml 2,750U/ml) の0.1ml と10mM トリス–HCl 緩衝液pH7.0 を0.4ml混合する。
基質溶液 (50mM オキサロ酢酸溶液)
オキサロ酢酸33mg を0.2M トリス–HCl 緩衝液pH9.0 5ml で溶解する。
酵素溶解希釈用液
10mM トリス–HCl 緩衝液pH8.0
試薬
TEA (トリエタノールアミン･塩酸塩) ：メルク製
塩化マンガン (MnCl₂ ・ 4H₂O) ：
富士フイルム和光純薬製　特級　#133–00725
NADH (ニコチンアミドアデニンジヌクレオチド・2Na・3H₂O・還元型) ：協和発酵製
LDH (乳酸脱水素酵素) ：ロシュ製　#10 127 876 001
オキサロ酢酸：
富士フイルム和光純薬製　#150-00411

酵素試料液

検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml とする。その液を酵素溶解希釈用液で0.5～1.4U/ml 濃度となるように適宜希釈する。

測定操作法

小試験管に反応試薬混合液2.0ml とLDH 溶液20 μl及び酵素試料液50 μl を正確に分注して25℃で予備加温する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液50μl を加える。
5 分経過後、基質溶液100 μl を加えて混和し、25℃で反応を開始する。
反応開始後、340nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
求められた吸光度変化を試料液はAs/min、盲検液はAb/min とする。
ΔA/min = (As/min−Ab/min) ≦ 0.15 Abs/min

計算

活性 (U/mg) = {(ΔA/min)/(6.22)} × 2.17/0.05 × 1/x

6.22 :	NADH の340nm におけるミリモル分子吸光係数 (cm² /μmole)
2.17 :	反応総液量 (ml)
0.05 :	反応に供した酵素試料液量 (ml)
X :	酵素試料液中の検品濃度 (mg/ml)

0.1 M TEA–NaOH buffer pH 8.0	0.90 ml
10 mM MnCl2 solution	0.10 ml
6.5 mM NADH solution	0.10 ml
Distilled water	0.90 ml

0.1M TEA–NaOH 緩衝液 pH8.0	0.90 ml
10mM 塩化マンガン溶液	0.10 ml
6.5mM NADH 溶液	0.10 ml
精製水	0.90 ml

Absorbance sample :	As/min
blank :	Ab/min

OXALOACETATE DECARBOXYLASE [OACⅡ] (T-209)