Enzymatic Assay of Diamine Oxidase (E.C. No. 1.4.3.22)

1. Objective

To standardize a procedure for the enzymatic assay of diamine oxidase.

2. Scope

This procedure applies to Product No. D7876, which has a specification for the enzymatic assay of Diamine Oxidase.

3. Definitions

3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

3.2. Unit Definition – One unit will oxidize 1.0 μmol of putrescine per hour at pH 7.2 at 37 ºC.

4. Discussion

Putrescine + H₂O + O₂   ^{Diamine Oxidase}   > 4 - Aminobutan aldehyde + NH₃ + H₂O₂

H₂O₂ + o - DIANISIDIN e(reduced)   ^Peroxidase   > 2H₂O + o - Dianisidin e(oxidized)

5. Responsibilities

Analytical services laboratory personnel should follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 37 °C, pH = 7.2, A_440nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric rate determination.

7.3 REAGENTS:

7.3.1     7.3.1. 100 mM Sodium Phosphate Buffer, pH 7.2 at 37 ºC (Buffer)
Prepare 12 mg/mL in purified water using sodium phosphate monobasic (Product No. S0751). Adjust to pH 7.2 at 37 ºC with 1 M NaOH.

7.3.2     75 mM Putrescine solution (Put)
Prepare 12.1 mg/mL  in reagent 7.3.1 using putrescine hydrochloride (Product No. P7505).

7.3.3     16 mM o-Dianisidine solution (ODA)
Prepare 5 mg/mL using o-Dianisidine (Product No. F5803) in purified water. Prepare fresh and protect from light.

7.3.3.1    o-Dianisidine tablets (Product No. D9154) is not suitable for this assay.

7.3.3.2    o-Dianisidine is a known carcinogen, handle with care.

7.3.4.    Peroxidase Enzyme solution (POD)
Immediately before use prepare a solution containing 2000 pyrogallol units/mL in cold reagent 7.3.1(Buffer), using Peroxidase Type II from horseradish (Product No. Number P8250).

7.3.5    Diamine Oxidase Enzyme solution (Enzyme)
Prepare a solution containing 3.0 to 6.0 units/mL of diamine oxidase in warm reagent 7.3.1 (37 ºC). Incubate for 30 minutes at 37 ºC before assaying to obtain complete enzyme dissolution.

7.4. PROCEDURE

7.4.1.    Pipette (in milliliters) the following reagents into suitable cuvettes:

	Test	Blank
Reagent 7.3.1 (Buffer)	2.50	2.60
Reagent 7.3.2 (Put)	0.20	0.20
Reagent 7.3.3 (ODA)	0.10	0.10
Reagent 7.3.4 (POD)	0.10	0.10

7.4.2.    Mix by inversion and equilibrate to 37 ºC. Monitor the A 440 nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Reagent 7.3.5 (Enzyme)

0.10

----

7.4.3.    Immediately mix by inversion and record the increase in A 440 nm/min for 10 minutes.

7.4.4.    Obtain the maximum linear rate for both the Test and the Blank using a minimum of a 1.0 minute period and four A440 nm points.

7.5 CALCULATIONS

7.5.1

3.0 = volume (in milliliters) of assay
60 = conversion factor from units/min to units/hour (Unit definition)
df = Dilution factor
11.3 = Millimolar extinction coefficient of oxidized o-Dianisidine at 440 nm
0.1 = Volume (in milliliters) of enzyme used

7.5.2

7.5.3

7.6. FINAL ASSAY CONCENTRATION
In a 3.0 mL reaction mix, the final concentrations are 96.7 mM sodium phosphate, 4.98 mM putrescine, 0.53 mM o-dianisidine, 200 units peroxidase, and 0.3-0.6 units diamine oxidase.

8. References 

(1964) Nature 204, 1195.