Hydrolyzed creatinine, used in the development and bulk formulation of enzymatic creatinine reagents.
Enzymatic properties
Source: Microorganism
Enzyme Committee No. : EC 3.5.2.10
Molecular weight: 29 kDa (SDS-PAGE)
Isoelectric point: 5.3
Km value: 5.0× 10-2 M (Creatinine),
8.0× 10-2 M (Creatine)
Inhibitors: Hg2+, Cu2+, Fe3+
Optimal pH: 7.0-8.0 Figure 1
Optimum temperature: 65℃ Figure 2
pH stability: pH 5.5-10.0 (25℃, 16 h) Figure 3
Thermal stability: stable below 65℃ (pH 8.0, 30 min) Figure 4
Stability: -25 ~ -15℃ standing storage
Maintain over 90% activity for 12 months Figure 5
Assay method for activity
1. Principle
2. Definition of enzyme activity
Unit enzyme activity is defined as the amount of enzyme required to catalyze a reaction to produce 1μmol creatine per minute under the following conditions.
3. Reagent preparation
Reagent I: 0.3M potassium phosphate buffer, pH 6.5.
Reagent II: 0.1M creatinine solution (1.13g creatinine dissolved in 100mL UP water).
Reagent III: 4% Na2CO3 solution (4.0g Na2CO3 dissolved in 100mL UP water).
Reagent IV: 2% alpha-naphthol solution (2.0g alpha-naphthol dissolved in 100 mL of 99.5% ethanol).
Reagent V: 1.2g NaOH and 3.2g Na2CO3 were dissolved in double steaming water at a constant volume of 100 mL.
Reagent VI: 0.05% diacetyl solution (0.05mL diacetyl with water to 100 mL).
Enzyme diluent: 5 mM Tris-HCl pH 8.0
4. Operation procedure
4.1. Add 0.1 mL reagent I and 0.8mL reagent II into a 5 mL centrifuge tube.
4.2. Water bath at 37℃ for 5 minutes.
4.3. Add 0.1 mL of the sample to be tested and incubate at 37℃ for 10 minutes.
4.4. Add 2.0mL reagent III to terminate the reaction, remove and place on ice.
4.5. Add the following reagents in the new 5 mL centrifuge tube in order:
The solution for step 4: 80 μL
Double steaming water: 720 μL
Reagent IV: 400 μL
Reagent V: 400 μL
Reagent VI: 400 μL
4.6. After standing at 25℃ for 1 h, add 2 mL of double steaming water to dilute.
4.7. Use a spectrophotometer to measure the absorbance at 525 nm.
* Replace enzyme liquid with enzyme diluent, other steps are the same, the absorbance of the resulting solution is blank absorbance (∆Ab)
∆A=∆As- ∆Ab
5. Vitality computing
Vt: Total volume of reaction liquid (1.0mL);
Vs: Enzyme liquid volume (0.1mL);
t: Reaction time (10 minutes);
df: Dilution ratio;
C: Enzyme concentration (mg/mL);
1.0: Optical path length (cm);
0.0704: Millimolar absorption coefficient (cm²/μmol) of chromophore at 525nm under standard reaction conditions.
ZJ25F0421092