Assay Procedure for Creatininase
Creatininase (Product No. C3172) from Pseudomonas sp. is a homohexameric enzyme with a molecular mass of 28.4 kDa per subunit. It is a cyclic amidohydrolase catalyzing the reversible conversion of creatinine to creatine. Each monomer contains a binuclear zinc centre near the C termini of the β-strands and the N termini of the main α-helices. These zinc ions indicate the location of the active site.
Assay Principle
The appearance of creatinine-picrate (orange dye based on Jaffe's reaction) is measured at 520 nm by spectrophotometry.
Unit definition
One unit causes the formation of one micromole of orange dye per minute under the conditions described below.
Reagents
Procedure
1. Pipette 1 mL of the substrate solution (A) into a test tube and equilibrate at 37 ℃ for about 5 minutes.
2. Add 0.1 mL of the enzyme solution* and mix.
3. After exactly 10 minutes at 37℃, immediately transfer an aliquot (0.1 mL)(1/11 volume) of the reaction solution to
2.0 mL of NaOH solution (B).
4. Add 1.0 mL of picric acid solution (C) and incubate at 25 ℃ for 20 minutes.
5. Measure the optical density at 520 nm against water (OD test).
At the same time, prepare the blank by transferring an aliquot (0.1 mL) of the reaction solution into NaOH solution (2.0 mL) 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 50 mM phosphate buffer, pH 7.5 and dilute to 1.8-2.4 U/mL with the same buffer, immediately before assay.
Calculation
Activity can be calculated by using the following formula：
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