Assay Procedure for Creatinase

Principle

The appearance of yellow dye formed by condensation of urea and p-dimethylaminobenzaldehyde (DAB) (Ehrlich reaction) is measured at 435nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of yellow dye per minute under the conditions described below.

Method

Reagents

 

A. Creatine solution
:0.1M[1.49g creatine /100ml of 50mM phosphate buffer, pH 7.5] (Should be prepared fresh)
B. DAB solution :Dissolve 2.0g of DAB in 100ml of dimethylsulfoxide and, to this solution, add 15ml of conc. HCl solution.
C. 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.
Concentration in assay mixture
Phosphate buffer 50 mM
Creatine 90 mM
  1. Add 0.1ml of the enzyme solution* and mix.
  2. After exactly 10 minutes at 37℃, add 2.0ml of DAB solution (B) to stop the reaction.
  3. Incubate at 25℃ for 20 minutes.
  4. Measure the optical density at 435nm against water (OD test).

At the same time, prepare the blank by first mixing the substrate solution with 2.0ml of DAB solution after a 10 min-incubation at 37℃, followed by the addition of the enzyme solution, and carry out the same procedure as test (procedure 4 and 5)(OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 2.0-3.0 U/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 (3.1ml)
Vs :Sample volume (0.1ml)
0.321 :Millimolar extinction coefficient of yellow dye (F/micromole)
1.0 :Light path length (cm)
t :Reaction time (10 minutes)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

Materials

     

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

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