CREATININE DEIMINASE from Microorganism

CNI-311

PREPARATION and SPECIFICATION
Appearance White amorphous powder, lyophilized
Activity GradeⅢ 10U/mg-solid or more
(containing approx. 30% of stabilizer)
Contaminants Creatinine amidohydrolase ≤1.0×10⁻²%
Creatine amidinohydrolase ≤1.0×10⁻²%
Urease ≤1.0×10⁻²%
NADH oxidase ≤1.0×10⁻²%
NH4⁺ ≤1.0×10⁻²% µg/u
Stabilizer Mannitol
PROPERTIES
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx. 260,000
Isoelectric point 4.4
Michaelis constant 3.5×10⁻³M (Creatinine)
Structure 6 subunits per enzyme molecule
Inhibitors Ag⁺, Hg⁺⁺, o-phenanthroline,monoiodoacetate
Optimum pH 8.5-9.5(Fig.3)
Optimum temperature: 65-75℃(Fig.4)
pH Stability pH 7.0-11.0 (30℃, 20hr)(Fig.5)
Thermal stability  below 65℃ (pH 7.5, 1hr)(Fig.6)
Effect of various chemicals (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of creatinine when coupled with glutamate dehydrogenase (GTD-211, GTD-209, GTD-309) in clinical analysis.

ASSAY

Principle:

creatinine deiminase

Creatinine+HO+H⁺                                  ►N-Methylhydantoin+NH₄

GIDH

NH⁺+α-Ketoglutarate+NADPH               ►Glutamate+H₂O+NADP⁺


GIDH:Glutamate dehydrogenase(NADP dependent)[L-Glutamate:NADP oxidoreductase (deaminating), (EC 1.4.1.4.)]
The disappearance of NADPH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of ammonia (the oxidation of micromole of NADPH) per minute under
the conditions described below.

Method:

Reagents
A. Creatinine solution 50mM (565.5mg creatinine/100ml of 50mM K-phosphate buffer, pH7.5)
B. NADPH solution 3.0mM (27.2mg NADPH・Na・4HO/10ml of 50mM K-phosphate buffer, pH 7.5)(Should be prepared fresh)
C. α-Ketoglutarate solution 10mM (14.6mg α-Ketoglutaric acid/10ml of 50mM K-phosphate buffer, pH 7.5)(Should be prepared fresh)
D. GIDH solution ca.1,000U/ml[Dilute Toyobo GradeII (Tris-HCl buffer solution, free from ammonia to ca.1,000U/ml with HO.)]
E. Enzyme diluent 50mM K-phosphate buffer, pH 7.5

Procedure

Concentration in assay mixture
K-Phosphate buffer 49 mM
Creatinine 38 mM
α-Ketoglutarate 0.95mM
NADPH
0.29mM
GIDH ca.16 µ/ml

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.

2.4 ml Substrate solution (A)
0.3ml NADPH solution (B)
0.3ml α-Ketoglutarate solution (C)
0.05ml GIDH solution (D)

2. Add 0.10ml of the enzyme solution* and mix by gentle inversion.

3. 2. Record the decrease in optical density at 340nm against water for 3〜4 minutes in a spectrophotometer
thermostated at 37°C, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution (ΔOD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E) and dilute to 0.15−0.4U/ml with the same buffer, immediately before assay.


Calculation

Activity can be calculated by using the following formula :

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

Volume activity (U/ml) =                                                               =ΔOD/min×5.06×df

6.22×1.0×Vs


Weight activity (U/mg) = (U/ml) × 1/C

Vt
: Total volume (3.15ml)
Vs
: Sample volume (0.10ml)
6.22
: Millimolar extinction coefficient of NADH(㎠/micromole)
1.0
: Light path length (cm)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. J.Szulmajster; Biochim.Biophys.Acta, 30, 154 (1958).
  2. T.Uwajima and O.Terada; Agr.Biol.Chem., 40, 1055 (1976).
  3. T.Uwajima and O.Terada; Agr.Biol.Chem., 41, 339 (1977).
  4. D.Tsuru; Rinsho Kensa, 22, 1331 (1978).
  5. T.W.Esders and S.Y.Lynn; J.Biol.Chem., 260, 3915 (1985).

Table 1. Effect of Various Chemicals on Creatinine deiminase
[The enzyme dissolved in 50mM Tris-HCl buffer, pH 7.5 (2U/ml) was incubated at 25℃ for 2hr with each chemical. The residual activity was assayed according to the routine method described above.]
Chemical Concn.(mM) Residual
activity(%)
Chemical Concn.(mM) Residual
activity(%)
None 100 MIA 1.0 0.6
Metal salt 1.0   NEM 1.0 86.1
MgCl₂
  103.8 IAA 1.0 60.1
CaCl₂   100.6 Hydroxylamine 1.0 86.1
Ba(OAc)₂ 105.7
EDTA 2.0 96.2
FeCl₃   5.0 o-Phenanthroline
1.0 0.7
CoCl₂   108.2 α,α′-Dipyridyl 0.1 100.6
MnCl₂   179.0 Borate 5.0 98.7
ZnSO₄   100.0 NaF 1.0 100.6
Cd(OAc)₂   143.0 NaN₃ 1.0 99.4
NiCl₂   103.2 TritonX-100 1.0% 95.6
CuSO₄   2.1 Brij 35 0.1% 83.5
Pb(OAc)₂   87.3 Span 20 0.5% 103.2
AgNO₃   1.1 Na-Cholate 0.5% 100.0
HgCl₂   1.6 SDS 0.5% 101.0
2-Mercaptoethanol 1.0 96.8 DAC 0.5% 81.8
PCMB 0.1 91.1      

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

 

To get a quote, contact us at info@toyobousa.com, or INQUIRY.