(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-47REACH適合品

LACTATE OXIDASE [LOXⅡ]

from Aerococcus viridans
(L–Lactate: oxygen oxidoreductase, EC 1.1.3.2)

L–Lactate + O₂ → Pyruvate + H₂O₂

Preparation and Specification

Appearance: : Yellowish amorphous powder, lyophilized

Specific activity: : More than 20 U/mg solid

Contaminants: :

POP: Less than 0.001 % (U/U)

GOD: Less than 0.001 % (U/U)

UODN: Less than 0.001 % (U/U)

CO: Less than 0.001 % (U/U)

Properties

Molecular weight: : 80 kDa (gel filtration)

Isoelectric point: : pH 4.6

Michaelis constants: : L–Lactate 7.0 × 10^-4M

Optimum pH: : 6.0–7.0Figure 1

pH stability: : 6.0–9.0 (50℃, 10 min) Figure 2

Optimum temperature: : 35℃

Thermal stability: : Stable at 50℃ and below ( pH 8.5, 10 min) Figure3

Storage stability: : At least one year at −20℃Figure4

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of lactic acid.

	LOX Ⅱ
L－Lactate + O₂	→	Pyruvate + H₂O₂
	POD
2 H₂O₂ + 4-AA + Phenol	→	Quinoneimine dye + 4 H₂O

Fig.1 pH Optimum

〇: Acetate buffer
△: Dimethyl glutarate-NaOH buffer
◇: KH₂PO₄-K₂HPO₄ buffer
●: Tris-HCI buffer
□: Glycine-NaOH buffer

Fig.2 pH Stability

40 mM buffer, 50℃, 10 min.
〇: Acetate buffer
△: Dimethylglutarate-NaOH buffer
◇: KH₂PO₄-K₂HPO₄ buffer
●: Tris-HCI buffer
□: Glycine-NaOH buffer

Fig.3 Thermal Stability

pH 8.5, 10 min.
40 mM Tris-HCI buffer

Fig.4 Storage (lyophilized powder)

－20℃

Assay

Principle

The assay is based on the increase in absorbance at 565 nm as the formation of quinoneimine dye proceeds in the following reactions:

	LOX Ⅱ
L–Lactate+O₂	→	Pyruvate+H₂O₂
	POD
2 H₂O₂+4–AA+DMA+H⁺	→	Quinoneimine dye +4 H₂O

DMA : N, N’–Dimethylaniline

Unit definition

One unit is defined as the amount of enzyme which generates 1 μmole of H₂O₂ per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Reaction mixture

0.2 M KH₂PO₄–NaOH buffer pH 6.5	0.2 ml
50 U/ml POD solution¹⁾	0.1 ml
15 mM 4–AA solution	0.1 ml
0.5 M DL–Lactic acid solution pH 6.5	0.1 ml
Distilled water	0.3 ml
Mix above reagents in advance. Just before measuring, add the reagent listed below and mix.
0.2% (W/V) DMA solution	0.2 ml

1) :	50 U/ml POD solution Dissolve 500 U (PPU) of POD with 10 ml of distilled water.

Reaction stopper
0.25% (W/V) LBS solution
LBS: Sodium lauryl sulfate
Enzyme dilution buffer
10 mM KH₂PO₄ –NaOH buffer pH 7.0 containing
10 μM FAD
FAD: Flavine adenine dinucleotide

Reagents
DL–Lactic acid:
FUJIFILM Wako Pure Chemical Corporation
Special grade　#128–00056
DMA: FUJIFILM Wako Pure Chemical Corporation
Special grade　#044–02763
FAD (2Na) : Kyowa Hakko Co., Ltd.
LBS: NACALAI TESQUE, INC. Extra pure #20123–22
4–AA: NACALAI TESQUE, INC.
Special grade　#01907–52
POD: Sigma Chemical Co.　Type Ⅱ　#P–8250

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 solution buffer to adjust the concentration as required.

Procedure

Pipette accurately 1.0 ml of reaction mixture into a small test tube and preincubate at 37℃.
After 5 min, add exactly 20 μl of enzyme solution and mix to start the reaction at 37℃.

※ In the case of a test blank, add 20 μl of enzyme dilution buffer in place of enzyme solution.
At 10 min after starting the reaction, add 2.0 ml of the reaction stopper to stop the reaction.
Measure the absorbance at 565 nm.

Absorbance sample : As

blank : Ab

△A = (As−Ab) ≦ 0.350 Abs

Calculation

Activity (U/mg of powder) = {(△ A/10)/(35.33×1/2)} × 3.02/0.02 × 1/x

35.33 :	millimolar extinction coefficient of quinoneimine dye at 565 nm ( cm² /μmole)
1/2 :	a multiplier derived from the fact that 2 mole of

	H₂O₂ produces 1 mole of quinoneimine dye
10 :	reaction time (min)
3.02 :	final volume (ml)
0.02 :	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. The enzyme activity will be retained for at least one year under this condition (Figure 4)

References

Eichel, H. J. and Rem, L. T. (1962) J. Biol. Chem., 237, 940–945.
Esders, T. W. and Goodhue, C. T. (1980) Eastman Kodak Company, U. S. Pat. 4,241,178.

LOX Ⅱ活性測定法 (Japanese)

試薬液

反応試薬混合液

0.2M KH₂PO₄–NaOH 緩衝液 pH6.5	0.2 ml
50U/ml POD 溶液 ¹⁾	0.1 ml
15mM 4–AA 溶液	0.1 ml
0.5M DL–乳酸溶液 pH6.5	0.1 ml
精製水を混合して置く。測定直前に前溶液と	0.3 ml
0.2% (V/V) DMA 溶液を混合する。	0.2 ml

1) ：	50U/ml POD 溶液 POD 500 単位 (PPU) を精製水10ml で溶解する。

反応停止液
0.25% (W/V) LBS 溶液
酵素溶解希釈用液
10 μM FAD を含む10mM KH₂PO₄–NaOH 緩衝液pH7.0
試薬
POD：シグマ製　Type Ⅱ　#P–8250
4–AA：ナカライテスク製　特級　#01907–52
乳酸 (DL–Lactic acid) ：
富士フイルム和光純薬製　特級　#128–00056
DMA (N,N’–ジメチルアニリン) ：
富士フイルム和光純薬製　特級　#044–02763
FAD (フラビンアデニンジヌクレオチド・2Na) ：
協和発酵製
LBS (ラウリルベンゼンスルホン酸ナトリウム) ：
ナカライテスク製　Extra pure #20123–22

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適宜希釈する。

測定操作法

小試験管に反応試薬混合液1.0ml を正確に分注して37℃で予備加温する。
5 分経過後、酵素試料液20 μ1 を加えて混和し、37℃で反応を開始する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液20μ1 を加える。
10 分経過後、反応停止液2.0ml を加えて混和し、反応を停止する。
565nm における吸光度を測定する
求められた吸光度を試料液はAs、盲検液はAb とする。
ΔA = (As−Ab) ≦ 0.350 Abs

計算

活性 (U/mg) = {(ΔA/10)/(35.33×1/2)} × 3.02/0.02 × 1/x

35.33 :	キノンイミン色素の565nm におけるミリモル分子吸光係数 (cm² / μmole)
1/2 :	H₂O₂ 2 モルからキノン色素1 モルが生成することによる係数
10 :	反応時間 (min)
3.02 :	反応総液量 (ml)
0.02 :	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)

Absorbance sample :	As
blank :	Ab

LACTATE OXIDASE [LOXⅡ] (T-47)