CREATININE DEIMINASE - Toyobo

CNI-311
TOYOBO ENZYMES
(Diagnostic Reagent Grade)
CREATININE DEIMINASE
from Microorganism
Creatinine iminohydrolase (EC 3.5.4.21) 15)
O
HN
H 2 O
HN N CH 3
Creatinine
O
HN
NH 4
O N CH 3
N-Methylhydantoin
PREPARATION and SPECIFICATION
Appearance
: White amorphous powder, lyophilized
Activity
: Grade10U/mg-solid or more
(containing approx. 30% of stabilizer)
Contaminants : Creatinine amidohydrolase ≤1.010 2 %
Creatine amidinohydrolase ≤1.010 2 %
Urease ≤1.010 2 %
NADH oxidase ≤1.010 2 %
NH 4 ≤1.010 2 g/u
Stabilizer
: Mannitol
PROPERTIES
Stability : Stable at 20 for at least one year Fig.1
Molecular weight : approx. 260,000
Isoelectric point : 4.4
Michaelis constant
: 3.510 3 M (Creatinine)
Structure
: 6 subunits per mol of enzyme
Inhibitors
: Ag ,Hg , o-phenanthroline,monoiodoacetate
Optimum pH : 8.59.5 Fig.2
Optimum temperature : 6575 Fig.3
pH Stability : pH 7.011.0 (30, 20hr) Fig.4
Thermal stability : below 65 (pH 7.5, 1hr) Fig.5
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.
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CNI-311
ASSAY
Principle:
CreatinineH 2 OH
creatinine deiminase
NH 4 -KetoglutarateNADPH
GIDH
N-MethylhydantoinNH 4
GlutamateH 2 ONADP
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 NADPHNa 4 4H 2 O/10ml of 50mM K-phosphate buffer, pH
7.5)(Should be prepared fresh)
C. -Ketoglutarate solution 10mM (14.6mg -Ketoglutaric acid/10ml of 50mM K-phosphate buffer, pH
7.5)(Should be prepared fresh)
D. GIDH solution
ca.1,000U/mlDilute Toyobo Grade (Tris-HCl buffer solution, free from ammonia
to ca.1,000U/ml with H 2 O.)
E. Enzyme diluent 50mM K-phosphate buffer, pH 7.5
Procedure
1. Prepare the following reaction mixture in a cuvette (d1.0cm)
Concentration in assay mixture
and equilibrate at 37 for about 5 minutes.
K-Phosphate buffer 49 mM
2.4 ml Substrate solution (A) Creatinine
38 mM
0.3 ml NADPH solution (B) -Ketoglutarate
0.95mM
NADPH
0.3 ml -Ketoglutarate solution (C)
0.29mM
GIDH
ca.16 u/ml
0.05ml GIDH solution (D)
2. Add 0.10ml of the enzyme solution and mix by gentle inversion.
3. Record the decrease in optical density at 340nm against water for 34 minutes in a spectrophotometer
thermostated at 37, and calculate the OD per minute from the initial linear portion of the curve (OD
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 (OD blank).
Dissolve the enzyme preparation in ice-cold enzyme diluent (E) and dilute to 0.150.4U/ml with the same
buffer, immediately before assay.
Calculation
Activity can be calculated by using the following formula
Volume activity (U/ml)
OD/min (OD testOD blank)Vtdf
6.221.0Vs
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 (F/micromole)
1.0 Light path length (cm)
df Dilution factor
C Enzyme concentration in dissolution (c mg/ml)
OD/min5.06df
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).
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CNI-311
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
None
Metal salt
MgCl 2
CaCl 2
Ba(OAc) 2
FeCl 3
CoCl 2
MnCl 2
ZnSO 4
Cd(OAc) 2
NiCl 2
CuSO 4
Pb(OAc) 2
AgNO 3
HgCl 2
2-Mercaptoethanol
PCMB
Residual
Concn.(mM)
activity
100%
1.0
1.0
0.1
103.8
100.6
105.7
5.0
108.2
179.0
100.0
143.0
103.2
2.1
87.3
1.1
1.6
96.8
91.1
Chemical
MIA
NEM
IAA
Hydroxylamine
EDTA
o-Phenanthroline
,-Dipyridyl
Borate
NaF
NaN 3
TritonX-100
Brij 35
Span 20
Na-Cholate
SDS
DAC
Concn.(mM)
1.0
1.0
1.0
1.0
2.0
1.0
0.1
5.0
1.0
1.0
1.0%
0.1%
0.5%
0.5%
0.5%
0.5%
Residual
activity
0.6
86.1
60.1
86.1
96.2
0.7
100.6
98.7
100.6
99.4
95.6
83.5
103.2
100.0
101.0
81.8
Ac, CH 3 CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide;
EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethyl-benzylalkyl-ammonium-chloride.
100
100
100
Residual Activity,%
50
-20
4
30
27
Relative Activity
50
Residual Activity,%
50
0 1 2 3 4 5 6 12
0 6 7 8 9 10
Period (months)
pH
0 4 5 6 7 8 9 10 11 12
pH
Fig.1. Stability (Powder form)
kept under dry conditions
Fig.2. pH-Activity
37in Britton-Robinson buffer; The
activity was assayed by the indophenol
method. 3)
Fig.4. pH-Stability
3020hr-treatment with Britton-Robinson
buffer
100
100
Relative Activity
50
Residual Activity,%
50
0 30 40 50 60 70 80 90
0 40 50 60 70 80
Temperature,
Temperature,
Fig.3.Temperature activity
Fig.5. Thermal stability
in 50mM K-phosphate buffer, pH 7.5; The
acitivity was assayed by the indophenol
method. 3)
1hr-treatment with 50mM K-phosphate
buffer, pH7.5
79

CNI-311
2 creatinine deiminase
4
GIDH
4
2
P
4
2
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
80