CHOLINE OXIDASE from Alcaligenes sp.

CHO-301

Choline: oxygen 1-oxidoreductase (EC 1.1.3.17)¹ ˜ ⁷⁾
(CH₃)₃N⁺CH₂CH₂OH + H₂O +2O₂                         ► (CH₃)₃N⁺CH₂COO‾ + 2H₂O₂

Choline                                                                                              Betaine

 

PREPARATION and SPECIFICATION
Appearance:  Yellowish amorphous powder, lyophilized
Activity:  GradeIII 10U/mg -solid or more
(containing approx. 20% of stabilizers)
Contaminant:  Catalase ≤1.0×10²%
Stabilizers:  EDTA, BSA, amino acids (glycine, sodium glutamate, etc.)
PROPERTIES
Stability:  Stable at -20°C for at least One year (Fig.1)
Molecular weight:  approx. 95,000
Isoelectric point:  4.1±0.1
Michaelis constants:  2.84×10⁻³M (Choline), 5.33×10⁻³M(Betaine aldehyde)
Structure:  One mol of FAD is covalently bound to mol of the enzyme⁸⁾
Inhibitors:  p-Chloromercuribenzoate, Cu⁺⁺, Co⁺⁺, Hg⁺⁺, Ag⁺
Optimum pH:  8.0-8.5(Fig.4)
Optimum temperature: 40-45℃(Fig.5)
pH Stability:  pH 7.0-9.0 (30°C, 2 hr)(Fig.6)
Thermal stability:   below 37℃ (pH 7.5, 10min)(Fig.7)
Effect of various chemicals:  (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of phospholipids when coupled with phospholipase D and for choline esterase-activity in clinical analysis.⁹~¹¹)

ASSAY

Principle:

choline oxidase

Choline+H₂O+2O₂                                  ►Betaine+2H₂O₂

peroxidase

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

The appearance of quinoneimine dye 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. Choline chloride solution:  2.1%[2.1g choline chloride/100ml of Tris-HCl buffer (D)](Should be prepared fresh)
B. 4-AA solution:  1.0% (1.0g 4-aminoantipyrine/100ml of H₂O)(Store at 4℃ in a brownish bottle)
C. Phenol solution:  1.0% (1.0g phenol/100ml of H₂O)(Store at 4℃ in a brownish bottle)
D. Tris-HCl buffer:  0.1M Tris-HCl buffer, pH 8.0[Dissolve 12.1g of Tris (MW=121.14) in ca.800ml of H₂O and, after adjusting the pH to 8.0 at 25℃ with 2.0 N HCl, fill up to 1,000ml with H₂O.]
E. Enzyme diluent:  10mM Tris-HCl buffer, pH 8.0 contg. 2mM EDTA and 1.0% KCl.

Procedure

Concentration in assay mixture
Tris buffer 97 mM
Choline chloride 0.14 M
EDTA 33 µM
KCI 2.2 mM
4-Aminoantipyrine 0.48 mM
Phenol 2.1 mM
POD ca.4.92U/ml

1. Prepare the following working solution (100ml) in a brownish bottle and store on ice.

97 ml Substrate solution (A)
1.0ml 4-AA solution (B)
2.0ml Phenol solution (C)
5.0mg Peroxidase from horseradish (110 purpurogallin units/mg)(Toyobo GradeIII)

2. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.

3.Add 0.05ml of the enzyme solution* and mix by gentle inversion.

4. Record the increase in optical density at 500nm against the working solution for 3 to 4 minutes in a
spectrophotometer thermostated at 37℃, and calculate theΔOD per minute from the initial linear portion of the curve.

* Dissolve the enzyme preparation in ice-cold Tris-HCl buffer (D) and dilute to 0.1-0.5U/ml with enzyme diluent (E).

Calculation

Activity can be calculated by using the following formula :

ΔOD/min×Vt×df

Volume activity (U/ml) =                                =ΔOD/min×10.17×df

12.0×1/2×1.0×Vs

 

Weight activity (U/mg) = (U/ml) × 1/C

Vt
: Total volume (3.05ml)
Vs
: Sample volume (0.05ml)
12.0
: 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. P.J.G Mann and J.H.Quastel; Biochem.J.,31, 869 (1937).
  2. P.J.G Mann et al; Biochem.J.,32, 1024 (1938).
  3. H.S.Shieh; Can.J.Microbiol., 10, 837 (1964).
  4. H.S.Shieh; Ibid.,11, 375 (1965).
  5. G.J.J.Kortstee; Arch.Mikirobiol., 71, 235 (1970).
  6. S.Ikuta, K.Matuura, S.Imamura, H.Misaki, Y.Horiuti; J.Biochem., 82, 157 (1977).
  7. S.Ikuta, S.Imamura, H.Misaki and Y.Horiuti ; J.Biochem., 82, 1741 (1977).
  8. M.Ohta-Fukuyama, Y.Miyake, S.Emi and T.Yamano; J.Biochem., 88, 197 (1980).
  9. M.Takayama et al; Clin. Chim. Acta, 79, 93 (1977).
  10. K.Sugawara and A.Kihara; Eisei Kensa, 27(1), 106 (1978).
  11. H.Okabe et al ; Clin.Chim.Acta, 80, 87 (1977).

Table 1. Effect of Various Chemicals on Choline oxidase
[The enzyme dissolved in 10mM Tris-HCl buffer, pH 8.0 contg. 2mM EDTA and 1.0% KCl (5U/ml) was incubated with each chemical at 25℃ for 1hr.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 MIA 2.0 87
Metal salt 2.0   NEM 2.0 100
MgCl₂   87 IAA 2.0 95
CaCl₂   92 Hydroxylamine 2.0 77
Ba(OAc)₂ 89 EDTA 5.0 92
FeCl₃   87 o-Phenanthroline 2.0 90
CoCl₂   89 α,α′-Dipyridyl 1.0 91
MnCl₂   91 Borate
50 94
ZnCl₂   88 NaF 2.0 92
CdCl₂   92 NaN₃ 2.0 92
NiCl₂   91 Triton X-100 0.10% 96
CuSO₄   92 Brij 35 0.10% 92
Pb(OAc)₂   87 Tween 20 0.10% 95
AgNO₃   80 Span 20 0.10% 94
HgCl₂   48 Na-cholate 0.10% 96
2-Mercaptoethanol 2.0 90 SDS 0.05% 95
PCMB 1.0 13 DAC 0.05% 91

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

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