Appearance Bluish green amorphous powder, lyophilized
Activity GradeⅢ 3,000U/mg-solid or more
Contaminants Catalase ≤1.0×10-2%


Stability Stable at −20℃ for at least one year (Fig.1)
(A decrease in activity of ca.10% may occur within 6 months)
Molecular weight 32,000 1)
Isoelectric point 4.95 2)
Structure 2 subunits per enzyme molecule (Each one mole of copper and zinc is bound to each subunit)
Inhibitors Cyanide 4),diethyldithiocarbamate 5)
Optimum pH 9.0 (Fig.4)
Optimum temperature 30℃ (Fig.5)
pH Stability pH 7.0−8.5 (25℃, 20hr) (Fig.6)
Thermal stability below 70℃ (pH 7.0, 30min)(Fig.7)




The appearance of reduced cytochrome C is measured at 550nm by spectrophotometry.

Unit definition

One unit causes half a maximum inhibition of cytochrome C reduction under the conditions described below.



A. K-Phosphate buffer, pH 7.8 75mM
B. EDTA solution 1.5mM Ethylenediaminetetraacetate・Na2
C. Xanthine solution 0.75mM (Dissolved in 0.004N NaOH solution)(Should be prepared fresh)
D. Xanthine oxidase solution 0.04U/ml[Dilute xanthine oxidase (ammonium sulfate suspension, ca.4U/ml) to 0.04U/ml with H2O](Should be prepared fresh)
E. Cytochrome C solution 0.15mM (from horse heart)(Should be prepared fresh)
F. Enzyme diluent 10mM K-Phosphate buffer,pH 7.8


1.Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 25℃ for about 5 minutes.

2.0 ml Buffer solution (A)
0.20ml EDTA solution (B)
0.20ml Xanthine solution (C)
0.20ml Cytochrome C solution (E)
0.20ml Enzyme solution* (F)
Concentration in assay mixture
K-Phosphate buffer 51 mM
Xanthine 50 μM
Cytochrome C 10 μM
EDTA 0.10 mM
Xanthine oxidase 2.6 mU/ml

2.Add 0.2ml of xanthine oxidase solution (D) and mix by gentle inversion.

3.Record the increase in optical density at 550nm against water for 2 to 3 minutes in a spectrophotometer thermostated at 25℃, 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 (F) is added instead of the enzyme solution.

*Dissolve the enzyme preparation in ice-cold enzyme diluent (F) and dilute to 0.5−2.0U/ml with the same buffer and store on ice.


Activity can be calculated by using the following formula :


Weight activity (U/mg) = (U/ml)×1/C

Vs : Sample volume (0.2ml)
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/ml)


1)J.M.McCord and I.Fridovich; J.Biol.Chem, 244, 6049 (1969)

2)J.Bannister, W.Bannister and E.Wood; Eur.J.Biochem., 18, 178 (1971)

3)I.Fridovich; Advan.Enzymol., 41, 35 (1974)

4)C.O.Beauchamp and I.Fridovich; Biochim.Biophys.Acta., 317, 50 (1973)

5)R.E.Heikkila, F.S.Cabbat and G.Cohen; J.Biol.Chem., 251, 2182 (1976)

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (Powder form at 5℃)

    Fig.2. Stability (Powder form at 5℃)

    (kept under dry conditions)

  • Fig.3. Stability (Liquid form at 5℃)

    Fig.3. Stability (Liquid form at 5℃)

    buffer composition : 10mM Kphosphate buffer, pH7.0

  • Fig.4. pH-Activity

    Fig.4. pH-Activity

    25℃ in 50mM buffer solution: pH3.0-5.5, acetate; pH5.5-8.5, phosphate; pH8.5-9.0, Tris-HCI pH10.0,sodium carbonate

  • Fig.5. Temperature activity

    Fig.5. Temperature activity

    (in 50mM phosphate buffer, pH7.0)

  • Fig.6. pH-stability

    Fig.6. pH-stability

    25℃, 20hr-treatment with 50mM buffer solution: (see Fig. 3)

  • Fig.7. Thermal stability

    Fig.7. Thermal stability

    30min-treatment with 50mM phosphate buffer, pH7.0


1. 原理


還元型Cytochrome Cの生成量を550nmにおける吸光度の変化で測定する。


下記条件下でCytochrome Cの還元を50%阻害する酵素量を1単位(U)とする


  • 75mM K-リン酸緩衝液,pH7.8
  • 1.5mM EDTA水溶液〔55.8㎎のEthylenediaminetetraacetate-Na2(2H2O)を蒸留水100mlに溶解する〕
  • 0.75mM Xanthine溶液〔0.004N NaOH溶液に溶解する〕(用時調整)
  • Xanthine oxidase溶液〔硫安懸濁液(約4U/ml) を蒸留水で0.04U/mlに希釈する〕(用時調製)
  • 0.15mM Cytochrome C水溶液(馬心臓由来 Cytochrome Cを蒸留水に溶解する)(用時調整)

酵素溶液:酵素標品を予め氷冷した10mM K-リン酸緩 衝液,pH7.8で溶解し,同緩衝液で0.5~ 2.0U/mlに希釈して氷冷保存する。


1.下記反応混液をキュベット(d=1.0cm)に調製し,25℃ で約5分間予備加温する。

2.0ml K-リン酸緩衝液 (A)
0.2ml EDTA水溶液 (B)
0.2ml Xanthine溶液 (C)
0.2ml Cytochrome C水溶液 (E)
0.2ml 酵素溶液 (F)

2.Xanthine oxidase溶液(D)0.20mlを添加し,ゆるやかに混和後,水を対照に25℃に制御された分光光度計で550nmの吸光度変化を2~3分間記録し,その初期直線部分から1分間当りの吸光度変化を求める(Δ ODtest)。

3.盲検は反応混液①に酵素溶液の代わりに酵素希釈液(10mM K-リン酸緩衝液,pH7.8)を加え,上記同様に操作を行って,1分間当りの吸光度変化を求める(Δ ODblank)。



U/mg = U/ml×1/C
C : 溶解時の酵素濃度(c mg/ml)