CREATINE AMIDOHYDROLASE from Microorganism

CNH-211, 311

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅡ (-211) 450U/mg-solid or more
GradeⅢ (-311) 150U/mg-solid or more
Contaminants:  NADH oxidase ≤5.0×10⁻²%
Catalase ≤2.0%
Stabilizers:  Sucrose, BSA
PROPERTIES
Stability:  Stable at -20°C for at least One year (Fig.1,2)
Molecular weight:  approx. 175,000 ¹ ⁾
Isoelectric point:  4.7 ³ ⁾
Michaelis constants:  3.2×10⁻²M (Creatinine), 5.7×10⁻²M (Creatine)
Structure:  6 subunits per enzyme molecule (One zinc is bound to each subunit) ³ ⁾
Inhibitors:  Ag⁺, Hg⁺⁺, N-bromosuccinimide, EDTA
Optimum pH:  6.5-7.5(Fig.5)
Optimum temperature: 70℃(Fig.6)
pH Stability:  pH 7.5-9.0 (5℃, 16hr)(Fig.7)
Thermal stability:   below 70℃ (pH 7.5, 30min)(Fig.8)
Effect of various chemicals:  (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of creatinine when coupled with creatine amidinohydrolase (CRH-211, CRH-221, CRH-229) and sarcosine oxidase (SAO-351) in clinical analysis.

ASSAY

Principle:

creatinine amidohydrolase

Creatine                                 ►Creatinine+H₂O

OH‾

Creatinine+Picric acid               ►Orange dye


The appearance of creatinine-picrate (orange dye based on Jaffe's reaction) is measured at 520nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of orange 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. NaOH solution:  0.5N
C. Picric acid solution:  1.0% (W/V)
D. Enzyme diluent:  50mM phosphate buffer, pH 7.5

Procedure

Concentration in assay mixture
Phosphate buffer 45mM
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℃, immediately transfer an aliquot (0.1ml)(1/11 volume) of the reaction solution to 2.0ml of NaOH solution (B).

4. Add 1.0ml of picric acid solution (C) and incubate at 25℃ for 20 minutes.

5. Measure the optical density at 520nm against water (OD test).
At the same time, prepare the blank by transferring an aliquot (0.1ml) of the reaction solution into NaOH solution (2.0ml) just after the addition of the enzyme solution into ice-cold substrate solution, and carry out the same procedure as the test (procedure 4 and 5)(OD blank).

* Dissolve the enzyme preparation in ice-cold 50mM phosphate buffer, pH 7.5 and dilute to 1.8−2.4U/ml with the same buffer, immediately before assay.


Calculation

Activity can be calculated by using the following formula :

ΔOD (OD test−OD blank ) × Vt × 11× df

Volume activity (U/ml) =                                                               =ΔOD×7.33×df

4.65×1.0× t ×Vs

 

Weight activity (U/mg) = (U/ml) × 1/C

Vt
: Total volume (3.1ml)
Vs
: Sample volume (0.1ml)
4.65
: Millimolar extinction coefficient of creatinine-picrate (orange dye)(㎠/micromole)
11
: Volume of reaction solution (1.1ml)/Sampling volume (0.1ml)
1.0
: Light path length (cm)
t
: Reaction time (10 minutes)
df
: Dilution factor of enzyme solution
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. D.Tsuru; Nucleic Acid and Amino Acids, 35, 31 (1977).
  2. D.Tsuru; Rinsho Kensa, 22, 1331 (1978).
  3. K.Rikitake, I.Oka, M.Ando, T.Yoshimoto and D.Tsuru; J.Biochem., 86, 1109 (1979).
  4. K.Yamamoto, M.Oka, T.Kikuchi and S.Emi; Biosci. Biotech. Biochem., 59 (7), 1331 (1995).

Table 1. Effect of Various Metals on Creatinine amidohydrolase
[The enzyme dissolved in 50mM K-phosphate buffer, pH 7.5 was incubated with each metal (0.2mM) for 30 minutes at 25℃.]
Metal Residual activity(%) Metal Residual activity(%)
None 100 MgCl₂ 82.8
FeSO₄ 91.0 NiCl₂ 82.8
FeCl₃ 93.0 CoSO₄ 78.5
HgCl₂ 1.4 BaCl₂ 71.2
Zn(OAc)₂ 79.9 Pb(OAc)₂ 88.0
Cu(OAc)₂ 22.7 AgNO₃ 0
Ca(OAc)₂ 96.8 CdCl₂ 80.8
MnCl₂ 94.0    
　

Ac, CH₃CO

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