Enzymatic Assay of Acid Phosphatase (EC 188.8.131.52)
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3.1 3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2. Unit Definition – One unit will hydrolyze 1.0 μmole of p-nitrophenyl phosphate per minute at pH 4.8 at 37°C.
T=37°C, pH=4.8, A410nm, Light path=1 cm
Spectrophotometric Stop Rate Determination
7.3.1 90 mM Citrate Buffer, pH 4.8 at 37°C (Buffer)
Prepare a 26.5mg/ml solution in purified water using Citric Acid, Trisodium, Dihydrate, Sigma-Aldrich Product Number C7254. Adjust to pH 4.8 at 37oC with 1M NaOH/HCL.
7.3.2 15.2 mM P-Nitrophenyl Phosphate (PNPP)
Prepare a 5.64 mg /ml solution in purified water using p-Nitrophenyl Phosphate, Sigma-Aldrich Stock Number 104-0, also known as Sigma-Aldrich Product Number S6750.
7.3.3 100mM Sodium Hydroxide Solution (NaOH)
Prepare 200 ml by diluting 20 ml of 1.0 N Sodium Hydroxide, Sigma-Aldrich Product Number S2567 in purified water, using a 200 ml volumetric flask.
7.3.4 Acid Phosphatase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing 0.15-0.25 un/ml of Acid Phosphatase in cold purified water.
7.4 TEST METHOD
7.4.1 Pipette (in milliliters) the following reagents into suitable containers:
|Reagent 7.3.1 (Buffer)||0.50||0.50|
|Reagent 7.3.2 (PNPP)||0.50||0.50|
7.4.2 Mix by inversion and equilibrate to 37oC. Then add:
|Reagent 7.3.4 (Enzyme)||0.10||----|
7.4.3 Immediately mix by inversion and incubate at 37oC for exactly 10 minutes. Then add:
|Reagent 7.3.3 (NaOH)||4.00||4.00|
|Reagent 7.3.4 (Enzyme)||-----||0.10|
7.4.4 Mix by inversion and using a suitable spectrophotometer record the A410nm for both the Test and the Blank.
|7.5.1||Units/ml enzyme =||ΔA410nm Test - ΔA410nm Blank)*(5.1)*(df)|
5.1 = Total volume (in milliliters) of solution
df = Dilution factor
10 = Time of assay (in milliliters) as per the Unit Definition
18.3 = Millimolar extinction coefficient of p-Nitrophenol
0.1 = Volume (in milliliter) 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 1.10 ml reaction mix the final concentrations are 41 mM Citric Acid, 6.9 mM p-Nitrophenyl Phosphate, and 0.015-0.025 units of Acid Phosphatase.
8. REFERENCES & ATTACHMENTS
Bergmeyer, H.U., Gawehn, K., and Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer H.U.) Volume I, 2nd ed., 495-496, Academic Press, Inc., New York, NY
Replacing OP SSPNPP02.