Replacing OP SPPUTR01. New document number assigned to conform to current document numbering system.
To standardize a procedure for the enzymatic assay of Diamine Oxidase.
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.
Putrescine + H2O + O2 Diamine Oxidase > 4 - Aminobutan aldehyde + NH3 + H2O2
H2O2 + o - DIANISIDIN e(reduced) Peroxidase > 2H2O + o - Dianisidin e(oxidized)
It is the responsibility of all Analytical Services laboratory personnel to follow this protocol as written.
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
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.
188.8.131.52 o-Dianisidine, Sigma-Aldrich Product Number D9154 (tablets), is not suitable for this assay.
184.108.40.206 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)
|Reagent 7.3.2 (Put)
|Reagent 7.3.3 (ODA)
|Reagent 7.3.4 (POD)
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.
||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
||Units/mg solid =
|mg solid/ml enzyme
||Units/mg protein =
|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.
8. REFERENCES & ATTACHMENTS
(1964) Nature 204, 1195.
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