CHOLESTEROL OXIDASE from Microorganism

COO-331

Cholesterol:oxygen oxidoreductase(EC 1.1.3.6)

PREPARATION and SPECIFICATION
Appearance:  Yellowish amorphous powder, lyophilized
Activity:  GradeIII 12U/mg -solid or more
Contaminants:  Catalase ≤1.0×10⁻¹%
Cholesterol esterase ≤1.0×10⁻²%
Stabilizers:  BSA, amino acids
PROPERTIES
Stability:  Stable at -20°C for at least One year (Fig.1)
Molecular weight:  approx. 55,000 (by gel-filtration)
Michaelis constants:  3.0×10⁻⁵M(Cholesterol)
Inhibitors:  Ionic detergents, Hg⁺⁺
Optimum pH:  7.0-8.0(Fig.2)
Optimum temperature: 60℃(Fig.3)
pH Stability:  pH 5.0-10.0 (25℃, 20hr)(Fig.4)
Thermal stability:   below 60℃ (pH 7.0, 15min)(Fig.5)
Substrate specificity:  (Table 1)
Effect of various chemicals:  (Table 2)

APPLICATIONS

This enzyme is useful for enzymatic determination of cholesterol in serum when coupled with cholesterol esterase (COE-301, COE-311, COE-313) in clinical analysis.

ASSAY

Principle:

cholesterol oxidase

Cholesterol +O₂                                  ►Cholest-4-en-3-one+H₂O₂

peroxidase

2H₂O₂+4-Aminoantipyrine+Phenol                                 ►Quioneimine dye+4H₂O

The appearance of quinoneimine dye formed when coupled with 4-aminoantipyrine and phenol is measured at 500nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per
minute under the conditions described below.

Method:

Reagents
A. 0.1M K-Phosphate buffer, pH 7.0
B. Cholesterol solution:  To 5.0ml of Triton X-100 on a hot plate or in a water bath, add 500mg of cholesterol and mix with a stirring bar until cholesterol dissolves. Add 90ml of distilled water to the hot cholesterol-Triton X-100 solution by slowly pouring along a stirring bar. Stir and allow to boil for 30 to 60 seconds. The solution will be cloudy. Cool under running water with gentle agitation, the solution will turn clear. Add 4.0g of sodium cholate and dissolve. Fill up the solution to 100ml with distilled water. This solution is stable for about one week at room temperature. If it becomes cloudy, warm slightly while stirring until it clears.
C. 4-AA solution:  1.76% (1.76g 4-aminoantipyrine/100ml of H₂O)
D. Phenol solution:  6.0% (6.0g phenol/100ml of H₂O)
E. POD solution:  Horseradish peroxidase 15,000 purpurogallin units/100ml of buffer (A)
F. Enzyme diluent:  20mM K-Phosphate buffer, pH 7.0 contg. 0.2% bovine serum albumin

Procedure

Concentration in assay mixture
K-Phosphate buffer 87 mM
Cholesterol 0.89mM
4-Aminoantipyrine 1.4 mM
Phenol 21 mM
Triton X-100 0.34 %
Sodium cholate 64 mM
BSA 33µg/ml
POD 5.U/ml

1. Prepare the following working solution (20 tests volume), immediately before use and store on ice in a brownish bottle.

51.0ml Buffer solution (A)
4.0ml Substrate solution (B)
1.0ml 4-AA solution (C)
2.0ml POD solution (E)

2. Pipette 2.9ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 3 minutes. Add 0.1ml of Phenol solution (D), mix and keep at 37℃ for another 2 minutes.

3.Add 0.1ml of the enzyme solution* and mix with gentle inversion.

4. Record the increase in optical density at 500nm against water for 3 to 4 minutes in a spectrophotometer
thermostated at 37°C, and calculate the ΔOD per minute from the initial 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 is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (F), and dilute to 0.1-0.3U/ml with the same buffer, and store on ice.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) × Vt × df

Volume activity (U/ml) =                                                               =ΔOD/min×4.499×df

13.78×1/2×1.0×Vs

 

Weight activity (U/mg) = (U/ml) × 1/C

Vt
: Total volume (3.1ml)
Vs
: Sample volume (0.1ml)
13.78
: Millimolar extinction coefficient of quinoneimine dye under the assay conditions(㎠/micromole)
1/2
: Factor based on the fact that one mole of H₂O₂ produces half a mole of quinoneimine dye
1.0
: Light path length (cm)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. W.Richmond; Clin.Chem., 19, 1350 (1973).
  2. H.M.Flegg; Ann.Clin.Biochem., 10, 79 (1973).
  3. C.C Alain et al; Clin.Chem., 20, 470 (1974).
  4. P.N.Tarbutton and C.R.Gunter; Clin.Chem., 20, 724 (1974).
  5. S.Nomoto; Rinsho Kensa, 20, 688 (1976).
  6. K.Kameno et al; Jap.J.Clin.Path., 24, 650 (1976).
  7. Y.Nishiya et al; Protein Engng,7, 231 (1997)

Table 1. Substrate Specificity of Cholesterol oxidase
Substrate(0.1mM) Relative activity(%) Substrate(0.1mM) Relative activity(%)
Cholesterol 100.0 Ergosterol 44.0
Pregnenolone 60.0 Lanosterol 1.6
ß-Cholestanol 60.0 Testosterone 1.0
ß-Sitosterol 120.0 Androsterone 1.5
Stigmasterol 34.0 Dehydroiso-androsterone 15.0
Table 2. Effect of Various Chemicals on Cholesterol oxidase
[The enzyme (1.0U/m) dissolved in 10mM phosphate buffer, pH 7.0 contg. 0.2% BSA(1.0U/ml) was incubated with each chemical at 25℃. for 1hr.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 NaF 20 100
Metal salt 2.0   NaN₃ 20 100
MgCl₂   100
 EDTA-2Na 5.0 100
CaCl₂   100 o-Phenanthroline 2.0 100
Ba(OAc)₂ 100 α,α'-Dipyridyl 1.0 100
FeCl₃   100 Borate 50 100
CoCl₂   100 IAA 2.0 100
MnCl₂   100 NEM 2.0 100
Zn(OAc)₂   100 Hydroxylamine 2.0 100
Cd(OAc)₂   100 2-Mercaptoethanol 2.0 100
NiCl₂   100 Triton X-100 0.10% 100
CuSO₄   90 Tween 20 0.10% 94
Pb(OAc)₂   100 Span 20 0.10% 90
HgCl₂   0 Na-cholate 0.10% 100
PCMB 2.0 100 SDS
 0.05% 100
MIA 2.0 100 DAC 0.05% 100

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, lodoacetamide; EDTA, Ethylenediamimetetraacetate;
SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

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