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Assay Procedure for Creatininase

Principle

The appearance of creatinine-picrate (orange dye based on Jaffe's reaction) is measured at 520nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of orange 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. NaOH solution :0.5N
C. Picric acid solution :1.0% (W/V)
D. 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 45 mM
Creatine 90 mM
  1. Add 0.1ml of the enzyme solution* and mix.
  2. After exactly 10 minutes at 37℃, immediately transfer an aliquot (0.1ml)(1/11 volume) of the reaction solution to 2.0ml of NaOH solution (B).
  3. Add 1.0ml of picric acid solution (C) and incubate at 25℃ for 20 minutes.
  4. Measure the optical density at 520nm against water (OD test).

At the same time, prepare the blank by transferring an aliquot (0.1ml) of the reaction solution into NaOH solution (2.0ml) just after the addition of the enzyme solution into ice-cold substrate solution, and carry out the same procedure as the test (procedure 4 and 5)(OD blank).

* Dissolve the enzyme preparation in ice-cold 50mM phosphate buffer, pH 7.5 and dilute to 1.8-2.4U/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 (3.1ml)
Vs :Sample volume (0.1ml)
4.65 :Millimolar extinction coefficient of creatinine-picrate (orange dye)(F/micromole)
11 :Volume of reaction solution (1.1ml)/Sampling volume (0.1ml)
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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