Creatinine Amidohydrolase catalyzes the hydrolytic reaction converting creatinine to creatine. The enzyme is purified from a microorganism. The molecular size of the enzyme is approximately 175,000. The enzyme is useful for the enzy-matic assay of creatinine when coupled with other related enzymes.
Reaction formula: Creatinine + H₂O → Creatine
PREPARATION and SPECIFICATION
| Appearance: | White amorphous powder, lyophilized |
| Activity: | ≥450 U/mg powder
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| Contaminants: | NADH oxidase ≤5.0×10⁻²%; Catalase ≤2.0% |
| Stabilizers: | Sucrose, BSA |
PROPERTIES
| Stability | Stable at -20℃ for at least one year | (Fig.1,2) | | Molecular weight | approx. 175,000 |
| | Isoelectric point | 4.7 |
| | Michaellis constants | 3.2×10⁻³M (Creatinine), 5.7×10⁻³M (Creatine) | | | Structure | 6 subunits per enzyme molecule (One zinc is bound to each subunit) |
| | 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 metals | - | (Table 1) |
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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:
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
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.
Concentration in assay mixture
Phosphate buffer 45mM
Creatine 90mM
Calculation
Activity can be calculated by using the following formula:
Vt: Total volume (3.1ml)
Vs: Sample volume (0.1ml)
4.65: Millimolar extinction coefficient of creatinine-picrate (orange dye) (cm²/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 (mg/ml)
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 | 80.0 | | MnCl₂ | 94.0 |
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Ac, CH₃CO.
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