PREPARATION and SPECIFICATION
This enzyme is useful for enzymatic determination of creatinine when coupled with creatinine amidohydrolase and creatine amidohydrolase.
One unit (U) is defined as the amount of enzyme which produces 1 μmol of H2O2 per min under the conditions described below.
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 – K2HPO4b uffer, pH 7.5.
Enzyme solution: Dilute the enzyme to 0.07 −0.17 U/ml with enzyme diluent.
REACTION MIXTURE (50 TESTS):
Reagent I 5 mL
Reagent II 10 mL
Reagent III 0.25 mL
Reagent IV 1.5 mL
Reagent V 1.5 mL
Pure H2O 31.75 mL
- Pipette 1 ml reaction mixture to a cuvette.
- Preincubate the reaction mixture at 37℃ for 5 min.
- 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
- N.Mori, M.Sato, Y.Tani and Y.Yamada; Agric.Biol.Chem., 44, 1391 (1980).
- M.Suzuki and M.Yoshida; Proceedings of the Symposium on Chemical Physiolosy and Pathology (Kyoto), Vol16, p.220 (1976).
- M.Suzuki; J. Biochem., 89, 599 (1981).