ACYL–CoA OXIDASE [ACOD] (T-17)

(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-17REACH適合品

ACYL–CoA OXIDASE [ACOD]

from Arthrobacter sp.
(Acyl–CoA: oxygen 2–oxidoreductase, EC 1.3.3.6)

Acyl–CoA + O2 trans– 2,3–dehydroacyl–CoA + H2O2

Preparation and Specification

Appearance
: Yellowish amorphous powder, lyophilized
Specific activity
: More than 20 U/mg solid

Properties

Substrate specificity
: See Table 1
Molecular weight
: 210 kDa (Sephadex G–150)
Isoelectric point
: pH 4.7
Michaelis constants
: Palmitoyl–CoA 2.0 × 10-5M
Optimum pH
: Serum Acyl–CoA 7.5
: Palmitoyl–CoA 8.5Figure 1
pH stability
: 6.0–7.5 (37℃, 60 min)Figure 2
Thermal stability
: Stable at 40℃ and below (pH 7.0, 10 min) Figure 3
Storage stability
Effects of various
: At least one year at −20℃Figure 4
chemicals
: See Table 2
Stabilizer
: FAD
Activator
: Triton X–100

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of fatty acid when coupled with Acyl–CoA synthetase (T–16).

ACS
FFA + CoA + ATP Acyl - CoA + PPi + AMP
ACOD
Acyl - CoA + O2 Enoyl - CoA + H2O2
POD
2 H2O2 + 4-AA + Phenol Quinoneimine dye + 4 H2O

FFA:Free fatty acid

 

Table 1. Substrate specificity of ACOD

Substrate Relative activity
(%)
Km value
(10- 5M)
Hexanoyl-CoA (6:0) 11  
Octanoyl-CoA (8:0) 60  
Decanoyl-CoA (10:0) 73  
Dodecanoyl-CoA (12:0) 87  
Tetradecanoyl-CoA (14:0) 99  
Hexadecanoyl-CoA (16:0) 53 2.0
Octadecanoyl-CoA (18:0) 16 3.8
Icosanoyl-CoA (20:0) 1  
9-Tetradecenoyl-CoA (14:1) 100  
9-Hexadecenoyl-CoA (16:1) 65  
9,12-Hexadecadienoyl- CoA (16:2) 55  
cis -9-Octadecenoyl-CoA (18:1) 45 4.0
trans -9-Octadecenoyl- CoA (18:1) 31  
d-12-Hydroxy-trans -9- octadecenoyl-CoA (18:1) 7  
cis -9, cis -12- Octadecenoyl-CoA (18:2) 31 3.8
cis -6, cis -9, cis -12- Octadecenoyl-CoA (18:3) 95 1.67
15-Tetracosenoyl-CoA (24:1) 7  

 

Table 2. Effect of various chemicals on ACOD activity

Additive Consentration Relative activity
(%)
None - 100
NaCl 1mM 102
KCl 1mM 104
LiCl 1mM 101
NH4Cl 1mM 101
MgCl2 1mM 138
BaCl2 1mM 147
CaCl2 1mM 137
MnCl2 1mM 155
ZnCl2 1mM 99
CoCl2 1mM 121
FeCl3 1mM 101
EDTA 1mM 74
Triton X-100 0.1% 140
Adekatol SO-120 0.1% 147
Sodium laurylbenzene sulfonate 0.1% 54
Sodium laurylsulfate 0.1% 43
Deoxycholate 0.1% 106

Fig.1 pH Optimum


pH 7 - 9 Tris-HCl buffer
〇: Palmitoyl-CoA
●: Serum Acyl-CoA

Fig.2 pH Stability


●: 3,3-Dimethylglutarate -NaOH
〇: Phosphate buffer
▲: Tris-HCl buffer

Fig.3 Thermal Stability


pH 7.0, 10 min.
Phosphate buffer

Fig.4 Storage (lyophilized powder)


〇: -20℃
□: 5℃

Assay

Principle
  1. The assay is based on the increase in absorbance at 500 nm as the formation of quinoneimine dye proceeds in the following reactions:

ACOD
Palmitoyl–CoA+O2 2–Hexadecenoyl–CoA+2HO2
 
POD
2H2O2+4–AA+Phenol Quinoneimine dye+4H2O
Unit definition
  1. 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
  1. Reaction mixture
    0.2 M Tris–HCl buffer pH 8.0 0.20 ml
    15 mM 4–AA solution 0.10 ml
    0.2% (W/V) Phenol solution 0.10 ml
    50 U/ml POD solution1) 0.10 ml
  1. 1% (W/V) Triton X–100 solution 0.10 ml
    5 mM Palmitoyl–CoA solution 2) 0.10 ml
    Distilled water 0.30 ml
    1) : 50 U/ml POD solution
    Dissolve 500 U (PPU) of POD with 10 ml of distilled water.
    2) : 5 mM Palmitoyl–CoA solution
    Dissolve 50.3 mg (purity calculation) of palmitoyl– CoA with 10 ml of 10 mM KH2PO4–NaOH buffer pH 7.0.
  2. Enzyme dilution buffer
    10 mM KH2PO4–NaOH buffer (pH 7.0) containing 3 mM ATP and 10 μM FAD
  3. Reagents
    4–AA: NACALAI TESQUE, INC. Special grade #01907–52
    POD: Sigma Chemical Co. Type Ⅱ # P–8250
    Triton X–100: The Dow Chemical Company
    Palmitoyl–CoA: Asahi Kasei Pharma Corporation
    ATP (2Na ・ 3H2O) : Kyowa Hakko Co., Ltd.
    FAD (2Na) : Kyowa Hakko Co., Ltd.
    ATP: Adenosine triphosphate
    FAD: Flavine adenine dinucleotide
Enzyme solution
  1. Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20ml. Dilute it with enzyme dilution buffer to adjust the concentration to within 0.2–0.5 U/ml.

Procedure
  1. Pipette accurately 1.0 ml of reaction mixture into a reaction cuvette (1 ml volume black cuvette) and preincubate at 37℃.
  2. After 5 min, add 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.
  3. After starting the reaction, measure the rate of increase in absorbance at 500 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) ≦ 0.060 Abs/min
Calculation
Activity (U/mg of powder) = {(△ A/min)/(12.0 × 1/2)} × 1.02/0.02 × 1/x
12.0 : millimolar extinction coefficient of quinoneimine dye at 500 nm (cm2 / μmole)
1/2 : a multiplier derived from the fact that 2 mol of H2O2 produce 1 mol of quinoneimine dye
1.02 : 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. Enzyme activity will be retained for at least one year under this condition (Figure 4) .

References
  1. Shimizu, S., Yasui, K., Tani, Y. and Yamada, H. (1979) Biochem. Biophys. Res. Commun., 91 (1) , 108–113.
  2. Kikuchi, T., Ogawa, M., Ando, M. and Nakagiri, Y (. 1979) Proceedings of Japanese Conference on Biochemistry of Lipids, 21, 144–147.
  3. Hosaka, K., Kikuchi, T, and Mitsuhida, N. (1979) Proceedings of the Symposium on Chemical Physiology, 19, 180.
  4. Shimizu, S., Inoue, K., Tani, Y. and Yamada, H. (1980) Anal. Biochem., 107, 193–198.
  5. Hosaka, K., Kikuchi, T., Mitsuhida, N. and Kawaguchi, A. (1981) J. Biochem., 89, 1799–1803.
  6. Kawaguchi, A., Tsubotani, S., Seyama, Y., Yamakawa, T., Osumi, T., et al. (1980) J. Biochem., 88, 1481–1486.

ACOD 活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液

    0.2M トリス–HCl 緩衝液pH8.0 0.20 ml

    15mM 4–AA 溶液 0.10 ml

    0.2% (W/V) フェノール溶液 0.10 ml

    50U/ml POD 溶液 1) 0.10 ml

    1% (W/V) トリトン X–100 溶液 0.10 ml

    5mM パルミトイル–CoA 溶液 2) 0.10 ml

    精製水 0.30 ml

    1) : 50U/ml POD 溶液 POD500 単位 (PPU) を精製水10ml で溶解す る。
    2) : 5mM パルミトイル–CoA 溶液 パルミトイル–CoA 50.3mg (純度換算) を 10mM KH2PO4–NaOH 緩衝液pH7.0 10ml で溶 解する。
  2. 酵素溶解希釈用液

    3mM ATP と10 μM FAD を含む10mM KH2PO4– K2HPO4 緩衝液pH7.0

  3. 試薬

    4–AA:ナカライテスク製 特級 #01907–52

    POD:シグマ製 Type Ⅱ #P–8250

    トリトンX–100:Dow Chemical 製

    パルミトイル–CoA:旭化成ファーマ製

    ATP (アデノシン三リン酸・2Na ・3H2O) : 協和発酵製

    FAD (フラビンアデニンジヌクレオチド・2Na) : 協和発酵製

酵素試料液
  1. 検品約20mg を精密に量り、酵素溶解希釈用液に溶 解して全容20ml とする。 その液を酵素溶解希釈用液で0.2~0.5U/ml 濃度とな るように適宜希釈する。
測定操作法
  1. 反応セル (1ml 用ブラックセル) 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。
  2. 5 分経過後、酵素試料液20 μl 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20 μl を加える。
  3. 反応開始後、500nm 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) ≦ 0.060 Abs/min

計算
  1. 活性 (U/mg) = {(△ A/min)/12.0 × 1/2)} × 1.02/0.02 × 1/x
    12.0 : キノンイミン色素の500nm におけるミリモル分子 吸光係数 (cm2/ μmole)
    1/2 : H2O2 2 モルからキノンイミン色素1 モルが生成す ることによる係数
    1.02 : 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)