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Assay Procedure for Creatinine Deiminase Microbial

Principle

GIDH:Glutamate dehydrogenase(NADP+ dependent)[L-Glutamate:NADP oxidoreductase (deaminating), (EC 1.4.1.4.)]
The disappearance of NADPH is measured at 340nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of ammonia (the oxidation of micromole of NADPH) per minute under the conditions described below.

Method

Reagents

 

A. Creatinine solution :50mM (565.5mg creatinine/100ml of 50mM K-phosphate buffer, pH7.5)
B. NADPH solution :3.0mM (27.2mg NADPH・Na4・4H2O/10ml of 50mM K-phosphate buffer, pH 7.5)(Should be prepared fresh)
C. α-Ketoglutarate solution :10mM (14.6mg α-Ketoglutaric acid/10ml of 50mM K-phosphate buffer, pH 7.5)(Should be prepared fresh)
D. GIDH solution :ca.1,000U/ml[Dilute with H2O.]
E. Enzyme diluent :50mM K-phosphate buffer, pH 7.5

Procedure

  1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.

    2.4 ml Substrate solution (A)
    0.3 ml NADPH solution (B)
    0.3 ml α-Ketoglutarate solution (C)
    0.05ml GIDH solution (D)
Concentration in assay mixture
K-Phosphate buffer 49 mM
Creatinine 38 mM
α-Ketoglutarate 0.95mM
NADPH 0.29mM
GIDH ca.16 u/ml
  1. Add 0.10ml of the enzyme solution* and mix by gentle inversion.
  2. Record the decrease in optical density at 340nm against water for 3~4 minutes in a spectrophotometer thermostated at 37℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).

At the same time, measure the blank rate by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution (ΔOD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E) and dilute to 0.15-0.4U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (3.15ml)
Vs :Sample volume (0.10ml)
6.22 :Millimolar extinction coefficient of NADH (F/micromole)
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Materials

     
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