Creatine Amidinohydrolase

ASSAY PRINCIPLE

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

UNIT DEFINITION One unit (U) is defined as the amount of enzyme which produces 1 μmol of yellow dye per min under the conditions described below.

REAGENTS PREPARATION

Reagent I: 0.1M Creatine solution.

Reagent II: DAB solution: dissolve 2.0 g of PDAB in 100 ml of dimethylsulfoxide and add 15ml HCl solution.

Enzyme diluent: 50 mM NaH2PO4-Na2HPO4 buffer, pH 7.5.

Enzyme solution: dilute the enzyme to 2.0-3.0 U/ml with enzyme diluent.

PROCEDURE

1. Pipette 1.0 ml of reagent I into a test tube and equilibrate at 37 ℃ for about 5 minutes.

4. Add 0.1 ml the enzyme solution and mix.
5. After 10 minutes at 37 ℃, add 2.0 ml of reagent II to stop the reaction. 
6. Incubate at 25 ℃ for 20 minutes.

Measure the optical density As at 435 nm.

At the same time, prepare the blank by first mixing the substrate solution with 2.0 ml of DAB solution after 0 min-incubation at 37 ℃, followed by the addition of the enzyme solution, and carry out the same procedure as test (procedure 4 and 5) (Ab).

∆A = As – Ab

CALCULATION

𝐕𝐨𝐥𝐮𝐦𝐞 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 (𝐔⁄𝐦𝐋)

=∆𝐀 × 𝟑. 𝟏 × 𝐝𝐟 𝟎. 𝟑𝟐𝟏 × 𝟎. 𝟏 × 𝟏𝟎 × 𝟏. 𝟎

= ∆𝐀 × 𝟗. 𝟔𝟔 × 𝐝𝐟

𝐖𝐞𝐢𝐠𝐡𝐭 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 (𝐔⁄𝐦𝐠) = 𝐕𝐨𝐥𝐮𝐦𝐞 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 × 𝟏/𝐂

3.1: Total volume (mL)

0.1: Enzyme volume (mL)

1.0: Light path length (cm)

10: Reaction time (minutes)

df: Dilution factor

C: Enzyme concentration (mg/mL)

0.321: Millimolar extinction coefficient of yellow dye under 435nm (cm2/μmol)

REFERENCES

1. D. Tsuru; Nucleic Acid and Amino Acids, 35, 31 (1977).

2. T.Yoshimoto, I.Oka and D.Tsuru; Arch.

Biochem. Biophys., 177, 508 (1976).

3. T. Yoshimoto, I. Oka and D. Tsuru; J.

Biochem., 79, 1381 (1976).

4. D. Tsuru; Rinsho Kensa, 22, 1331 (1978).

This enzyme is useful for enzymatic determination of creatinine when coupled with creatinine amidohydrolase and sarcosine oxidase.