SOX (Sarcosine oxidase)
This enzyme is useful for enzymatic determination of creatinine when coupled with creatinine amidohydrolase and creatine amidohydrolase.
Specification
|
Appearance |
Yellowish amorphous powder, lyophilized |
|
Protein purity |
≥90% (from SDS-PAGE) |
|
Activity |
≥15 U/mg solid |
|
Catalase |
≤0.03% |
|
Creatinine amidohydrolase |
≤0.01% |
|
Creatine amidohydrolase |
≤0.01% |
|
ATPase |
≤ 0.005% |
Properties
|
EC number |
|
|
|
Molecularweight |
45 kDa (SDS-PAGE) |
|
|
Isoelectricpoint |
5.3 |
|
|
Michaelis constants |
5.6×10-3 M (Sarcosine) |
|
|
Inhibitors |
SDS, Zn2+, Cd2+ |
|
|
Optimum pH |
8.0 |
Fig. 1 |
|
Optimum temperature |
50℃ |
Fig. 2 |
|
pH stability |
pH 7.0 – 9.5 (25℃, 16 h) |
Fig. 3 |
|
Thermal stability |
Below 50 ℃ (pH 7.5, 30 min) |
Fig. 4 |
|
Storage stability |
At least one year at -25 ～- 15 ℃ |
Fig. 5 |
|
EC number |
|
|
|
Molecular weight |
45 kDa (SDS-PAGE) |
|
Figures
Assay Principle
The appearance of Quinoneimine dye is measured at 555 nm by spectrophotometry.
Unit definition
One unit (U) is defined as the amount of enzyme which produces 1 μmol of H2O2 per min under the conditions described below.
Reagents preparation
Reagent I: 0.2 M Tris-HCl buffer, pH 8.0.
Reagent II: 1.0 M Sarcosine solution.
Reagent III: 1 kU/mL POD solution.
Reagent IV: 50 mM 4-AA solution.
Reagent V: 50 mM TOOS solution.
Enzyme diluent: 10 mM KH2PO4 - K2HPO4 buffer, pH 7.5.
Enzyme solution: Dilute the enzyme to 0.07 −0. 17 U/ml with enzyme diluent.
Procedure
1. Pipette 1 ml reaction mixture to a cuvette.
2. Preincubate the reaction mixture at 37 ℃ for 5 min.
3. Add 20 ul the enzyme solution and mix to start the reaction, record ΔAs at 555 nm in 1 minute in a spectrophotometer.
Measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab
Calculation
1.02: Total volume (mL)
0.020: Enzyme volume (mL)
t：Reaction time (1min)
1.0: Light path length (cm)
1/2: 1 μmol of H2O2 produces 0.5 μmol of Quinoneimine dye
df: Dilution factor
C: Enzyme concentration (mg/mL)
39.2: Millimolar extinction coefficient of quinoneimine dye under 555 nm (cm2/μmol)
References
1. N.Mori, M.Sato, Y.Tani and Y.Yamada; Agric.Biol.Chem., 44, 1391 (1980).
2. M.Suzuki and M.Yoshida; Proceedings of the Symposium on Chemical Physiolosy and Pathology (Kyoto), Vol16, p.220 (1976).
3. M.Suzuki; J. Biochem., 89, 599 (1981).
