CREATINE AMIDINOHYDROLASE from Microorganism

CRH-221, 229

PREPARATION and SPECIFICATION
Appearance:  (-221) White amorphous powder, lyophilized
(-229) Clear solution
Activity:  GradeⅡ
(-221) 4.0U/mg-solid or more
(-229) 2000U/ml or more
Contaminants:  NADH oxidase ≤5.0×10⁻²%
Catalase ≤2.0%
Stabilizers:  Sugars, EDTA
PROPERTIES
Stability:  (-221) Stable at -20°C for at least One year (Fig.1)
(-229) Stable at 4°C
Molecular weight:  approx. 67,000 (by gel filtration)
Structure:  2 subunits per enzyme molecule
Isoelectric point:  4.5±0.1
Michaelis constant:  4.5×10⁻³ M (Creatine)
Inhibitors:  Hg⁺⁺, Cu⁺⁺, Ag⁺, SH reagent (NEM), PCMB
Optimum pH:  6.5-7.5(Fig.2)
Optimum temperature: 40-50℃(Fig.3)
pH Stability:  pH 4.0-10.0 (25℃, 20hr)(Fig.4)
Thermal stability:   below 50℃ (pH 7.5, 30min)(Fig.5)
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.

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] (Prepare freshly)
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 with each chemical at 25℃ for 30 minutes.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 NaF 1.0 99
Metal salt 1.0   PCMB 0.33 0
CaCl₂
   102 MIA 1.0 94
MnCl₂   96 IAA 1.0 100
MgCl₂ 103 NaN₃ 10 96
NiCl₂   104 o-Phenanthroline 1.0 99
CoCl₂   104 Hydroxylamine 1.0 99
Ba(OAc)₂   98 NEM 10 32
Cd(OAc)₂   98 Triton X-100 0.5% 99
FeCl₃   89 Brij 35 0.5% 94
FeSO₄   81 Tween 20 0.5% 98
HgCl₂   0 Span 20 0.5% 99
ZnSO₄   63 Na-cholate 0.25% 96
CuSO₄   0 SDS 0.25% 32
Pb(OAc)₂   98 DAC 0.5% 0
AgNO₃   0      
EDTA 20 98      
α,α′-Dipyridyl 1.0 102      

Ac, CH₃CO; PCMB, P-Chloromercuribenzoate; MIA, Monoidoacetate; EDTA, Ethylenediaminetetraacetate; IAA, lodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzlyalkylammonium chloride.

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