CREATINE AMIDINOHYDROLASE from Actinobacillus sp.

PREPARATION and SPECIFICATION
Appearance White amorphous powder, lyophilized
Activity GradeⅡ 6.0U/mg-solid or more
(containing approx. 50% of stabilizers)
Contaminants NADH oxidase ≤5.0×10⁻²%
Catalase ≤2.0%
Stabilizers Sugars, EDTA
PROPERTIES
Stability Stable at -20℃ in at least one year(Fig.1,2)
Molecular weight approx. 100,000
Isoelectric point 4.6±0.1
Michaelis constant 1.9×10⁻² M (Creatine)
Structure 2 subunits per enzyme molecule
Inhibitors Cu⁺⁺, Hg⁺⁺, Ag⁺
Optimum pH 8.0(Fig.4)
Optimum temperature: 40℃(Fig.5)
pH Stability pH 5.5-9.0 (25℃, 16hr)(Fig.6)
Thermal stability  below 50℃ (pH 7.5, 30min)(Fig.7)
Effect of various chemicals (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of creatine and creatinine when coupled with creatinine amidohydrolase (CNH-211, CNH-311) and sarcosine oxidase (SAO-351) in clinical analysis. ⁴⁾

CRH-211

ASSAY

Principle:



The appearance of yellow dye formed by condensation of urea and p-dimethylaminobenzaldehyde (DAB) (Ehrlich reaction) is measured at 435nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of yellow dye per minute under the conditions described
below.

Method:

Reagents
A. Creatine solution 0.1M[1.49g creatine (Nacalai tesque)/100ml of 50mM phosphate buffer, pH 7.5] (Should be prepared fresh).
B. DAB solution Dissolve 2.0g of DAB in 100ml of dimethylsulfoxide and, to this solution, add 15ml of conc. HCl solution.
C. Enzyme diluent 50mM Phosphate buffer, pH 7.5

Procedure

Concentration in assay mixture
Phosphate buffer 50mM
Creatine 90mM

1. Pipette 1.0ml of the substrate solution (A) into a test tube and equilibrate at 37℃ for about 5 minutes.

2. Add 0.1ml of the enzyme solution* and mix.

3. After exactly 10 minutes at 37℃, add 2.0ml of DAB solution (B) to stop the reaction.

4. Incubate at 25℃ for 20 minutes.

5. Measure the optical density at 435nm against water (OD test).
At the same time, prepare the blank by first mixing the substrate solution with 2.0ml of DAB solution after a 10 min-incubation at 37℃, followed by the addition of the enzyme solution, and carry out the same procedure as test (procedure 4 and 5)(OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 2.0-3.0 U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD (OD test−OD blank ) × Vt × df

Volume activity (U/ml) =                                                               =ΔOD×9.65×df

0.321×1.0× t ×Vs

 

Weight activity (U/mg) = (U/ml) × 1/C

Vt
: Total volume (3.1ml)
Vs
: Sample volume (0.1ml)
0.321
: Millimolar extinction coefficient of yellow dye(㎠/micromole)
1.0
: Light path length (cm)
t
: Reaction time (10 minutes)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. D.Tsuru; Nucleic Acid and Amino Acids, 35, 31 (1977).
  2. T.Yoshimoto, I.Oka and D.Tsuru; Arch.Biochem.Biophys., 177, 508 (1976).
  3. T.Yoshimoto ,I.Oka and D.Tsuru; J.Biochem., 79, 1381 (1976).
  4. D.Tsuru; Rinsho Kensa, 22, 1331 (1978).

Table 1. Effect of Various Chemicals on Creatine amidinohydrolase
[The enzyme dissolved in 50mM Tris-HCl buffer, pH 7.5 (80U/ml) was incubated at 25℃ for 30 minutes with each chemical.]
Chemical Concn.(mM) Residual
activity(%)
Chemical Concn.(mM) Residual
activity(%)
None 100 NaF 1.0 105
Metal salt 1.0   PCMB 0.33 3.3
CaCl₂
  107 MIA 1.0 106
MnCl₂   109 IAA 1.0 103
MgCl₂ 103 NaN 10 106
NiCl₂   107 o-Phenanthroline 1.0 108
CoCl₂   108 Hydroxylamine 1.0 105
Ba(OAc)₂   104
NEM 10 0.3
Cd(OAc)₂   88 Triton X-100 0.5% 94
FeCl₃   103 Brij 35 0.5% 103
FeSO₄   102
Tween 20 0.5% 100
HgCl₂   2.7
Span 20 0.5% 106
ZnSO₄   97
Na-cholate 0.5% 103
CuSO₄   11 SDS 0.25% 102
Pb(OAc)₂   108
DAC 0.5% 1.7
AgNO₃   2.5      
EDTA 20 99      
α,α′-Dipyridyl 1.0 100      

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

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