Manuals And Inserts
Specific Protocol
Phospholipase D Assay
I. PRINCIPLES OF THE ASSAY
The assay comprises three reaction steps. In the first step, phospholipase D catalyzes hydrolysis of phosphatidylcholine to choline and phosphatidic acid. In the second step, oxidation of choline catalyzed by choline oxidase produces two hydrogen peroxides. Peroxidase catalyzes the third step, in which the two hydrogen peroxides, phenol and 4-aminoantipyrine react to produce a quinoneimine dye, with a millimolar extinction coefficient of 12.2mM-1cm-1 at 500nm. Measurement of the absorbance at 500 nm can then be used to calculate the amount of choline produced by the phospholipase reaction. Both the choline oxidase and peroxidase reactions take place in the same solution after addition of ‘Reaction Mix II’. The addition of ‘Reaction Mix II’ also stops the phospholipase D reaction by addition of the chelator EDTA in excess of the calcium ion concentration. The procedure is written for use with a spectrophotometric plate reader and standard well 96-well microtiter plates. The final assay volume is 300 μl. With appropriate volume adjustments (1 or 3 ml final volume) reactions may also be read with standard or semi-micro cuvettes.
II. SOLUTIONS
Reaction Mix I (may be stored at –20°C): 44.4mM TRIS-HCl, pH8.0; 11.1mM CaCl2; 5.55mM L--phosphatidylcholine, dioleyl (C18:1, [cis]-9)/1.11% w/v Triton X-100 (added from a stock of 25mM phosphatidylcholine in 5% Triton X-100)
Phospholipase D (prepare day of assay and keep on ice): S. chromofuscus phospholipase D (BML-SE301) diluted to 4 to 40 units/ml with 10mM TRIS-HCl, pH 8.0, 0.1% Triton X-100, 0.5mg/ml BSA. A dilution of 20 units/ml, assayed as described below, will produce an A500nm of ~0.5.
Choline Solutions for Preparing Standard Curve (store at –20°C): Concentrations of choline as desired from 0 to 6mM in water.
Reaction Mix II (prepare fresh for assay and warm to 37°C just before use): 40mM TRIS-HCl pH 8.0; 2.4mM EDTA; 0.6mM 4-aminoantipyrine (added from 15mM stock, stored at –20°C); 0.008% w/v phenol (added from 0.2% stock stored at –20°C); 8 units/ml choline oxidase (Sigma Cat. No. C5896); 8 units/ml peroxidase (Sigma Cat. No. P8250).
Choline Chloride Standard Solutions (in water): For example, choline chloride solutions of 0.3, 0.6, 1.2, 1.8, 3.0, 4.8 and 6.0mM, will, respectively, produce final concentrations of 5, 10, 20, 30, 50, 80, and 100μM in the assay described below. For a 300μl final assay volume in a standard 96-well plate, the optical path-length will be 0.8 cm. Choline added to a final concentration of 0.1mM (100μM) will produce 0.1mM quinoneimine dye and an absorbance at 500nm of: 12.2mM-1cm-1 x 0.1mM x 0.8 cm = 0.976.
III. PROCEDURE
A. Using a standard 96-well microtiter plate (flat-bottom, polystyrene), add 45μl of ‘Reaction Mix I’ to all wells to be used in the assay (blank, standard or enzyme wells). Warm plate to 37°C.
B. Add 5μl of water to the wells that will serve as blanks. If preparing a choline standard curve, add 5μl of the choline chloride standard solutions to the wells devoted to that.
C. Initiate phospholipase D reactions by adding 5μl of diluted enzyme to the appropriate wells and mixing. Start timing 10 min.
D. Stop the phospholipase D reactions at 10 min. by addition of 250μl ‘Reaction Mix II’. Also add 250μl ‘Reaction Mix II’ to blank and choline standard wells.
E. Continue incubation at 37°C to allow the choline oxidase and peroxidase reactions to produce the colored reaction product. Color development should reach a maximum in 30-60 min., and will remain stable for at least 2-3 hr. afterwards.
F. Quantitate the choline produced by the phospholipase D by comparison to the standard curve or use of the extinction coefficient for the quinoneimine dye
μmol choline/well = (A500nm x 0.3ml)/(0.8cm x 12.2mM-1cm-1) or [choline] (mM) x 0.3ml
For a 10 min. reaction time, Phospholipase D Units/well = 6 x μmol choline/well
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