Enzymatic Assay of Diamine Oxidase (E.C. No. 18.104.22.168)
Replacing OP SPPUTR01. New document number assigned to conform to current document numbering system.
This procedure applies to Sigma-Aldrich Product Number D7876, which has a specification for the enzymatic assay of Diamine Oxidase.
3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2. Unit Definition – One unit will oxidize 1.0 μmole of Putrescine per hour at pH 7.2 at 37ºC.
T = 37°C, pH = 7.2, A440nm, Light path = 1 cm
Spectrophotometric rate determination.
7.3.1 7.3.1. 100 mM Sodium Phosphate Buffer, pH 7.2 at 37ºC (Buffer)
Prepare a 12 mg/ml solution in purified water using Sodium Phosphate Monobasic, Sigma-Aldrich Product Number S0751. Adjust to pH 7.2 at 37ºC with 1 M NaOH.
7.3.2 75 mM Putrescine solution (Put)
Prepare a 12.1 mg/ml solution in reagent 7.3.1 using Putrescine Hydrochloride, Sigma-Aldrich Product Number P7505.
7.3.3 16 mM o-Dianisidine solution (ODA)
Prepare a 5 mg/ml solution using o-Dianisidine, Sigma-Aldrich Product Number F5803, in purified water. Prepare fresh and protect from light.
22.214.171.124 o-Dianisidine, Sigma-Aldrich Product Number D9154 (tablets), is not suitable for this assay.
126.96.36.199 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, Sigma-Aldrich Product 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 (37ºC). Incubate for 30 min at 37ºC before assaying to obtain complete enzyme dissolution.
7.4.1. Pipette (in milliliters) the following reagents into suitable cuvettes:
|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 37ºC. Monitor the A 440nm until constant, using a suitably thermostatted spectrophotometer. Then add:
7.4.3. Immediately mix by inversion and record the increase in A 440nm/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 A440nm points.
|7.5.1||Units/ml enzyme =||(ΔA440nm / minTest - ΔA440nm / min Blank)*(60)*(3.0)*(df)|
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 m
0.1 = Volume (in milliliters) of enzyme used
|7.5.2||Units/mg solid =||units/ml enzyme|
|mg solid/ml enzyme|
|7.5.3||Units/mg protein =||units/ml enzyme|
|mg protein/ml enzyme|
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.
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