1. Introduction

Because of their critical functions in eukaryotic cells, methods for measuring protein phosphatases were established at least as early as 1953[1]. In 1965 Fernley and Walker[2] described the use of 4-methylumbelliferyl phosphate (MUP) as a substrate for alkaline phosphatase. Dephosphorylation of MUP yields a highly fluorescent and stable product: 4-methylumbelliferone (4MU).

An improved method involves the use of 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), which can assay both acid and alkaline phosphatase activity. The hydrolysis product of DiFMUP to DiF4MU exhibits both a lower pka (4.9 versus 7.8) and a higher fluorescence quantum yield (0.89 versus 0.63) than the hydrolysis product of MUP. The lower pka of its hydrolysis product makes DiFMUP a sensitive substrate for acid phosphatases, which is not possible with MUP because its flourescence must be measured at alkaline pH. Furthermore, with its high quantum yield, DiFMUP increases the sensitivity of both acid and alkaline phosphatase measurements. Lastly, fluorination reduces the susceptibility of the methylumbelliferone fluorophore to photobleaching effects without significantly affecting the extinction coefficient or excitation/emission maxima. Combined with theTurner BioSystems TD-700 Laboratory Fluorometer, DiFMUP enables researchers to quantitate as little as 1.0 pg/ml alkaline phosphatase.

2. Materials Required

• TD-700 Fluorometer with standard PMT and 10 mm × 10 mm cuvette adaptor (P/N 7000-009)
• Near UV Lamp (P/N 10-049)
• Excitation Filter, 365 nm (P/N 034-0365)
• Emission Filter, 410-610 nm (P/N 10-110R-C)
• 10 mm × 10 mm methylacrylate fluorescence cuvettes (P/N 7000-959)
• DiFMUP(6,8-difluoro-4- methylumbelliferyl phosphate, ammonium salt)
• Sodium Carbonate, Anhydrous (NA2CO3, MW=106.00)
• Alkaline Phosphatase Standard
• 50mM Tris Buffer pH 8.0.
• Bovine Serum Albumin (BSA)

3. Experiment Protocol

3.1 Reagent Preparation

DiFMUP Substrate Stock Solution, 1mg/mL. Dissolve 5.0 mg DiFMUP into 5.0 mL 50 mM Tris/0.1% BSA buffer, pH 8.0. Make up fresh daily.

NOTE: DiFMUP spontaneously hydrolyzes in aqueous solution. It should be stored in its solid form and made up just prior to use.

DiFMUP Substrate Working Solution, 10µg/mL. Dilute 300 µL DiFMUP Substrate Stock Solution into 30 mL 50mM Tris/0.1% BSA Buffer, pH 8.0. Make up fresh daily.

Alkaline Phosphatase Stock Solution, 1 mg/mL. (Biozyme calf-intestine alkaline phosphatase, 15.42 mg/ml). Dilute 100 µl alkaline phosphatase into 1.4 mL 50mM Tris/0.1% BSA buffer, pH 8.0.

Alkaline Phosphatase Standard Solution, 500 µg/mL. Dilute 1ml AP Stock Solution with 1mL Tris/0.1% BSA buffer.

Carbonate Stop Buffer(0.20M). Dissolve 21.2g Sodium Carbonate, anhydrous into 1000mL DI water.

3.2. Instrument Set-Up

1. Turn on the TD-700 Fluorometer. Allow it to warm up for 10 minutes (600 seconds).
2. Ensure the lamp is installed by checking that the small window in the back panel is lit or by removing the filter cylinder and observing the lamp emission in the sample chamber.
3. Ensure that the excitation filter, P/N 034-0365, is installed in the position marked EX and the emission filter, P/N 10-110R-C, is installed in the corresponding position marked EM in the filter cylinder.

3.3. Instrument Calibration

1. Calibrate the instrument with the 20µg/mL AP standard solution listed in Table 1 according to the TD-700 Operating Manual, page 21. Set the sample setting to 900. Choose [9] or [No] when prompted to subtract blank.

3.4 Alkaline Phosphatase Standard Curve

1. To generate a single-replicate, six- point standard curve from 20 µg/mL to 1 µg/mL, add 0.5 mL 10µg/mL DiFMUP Working Solution to each of 6 cuvettes.
2. Add an aliquot of 500 µg/mL AP Standard Solution to a cuvette and incubate the mixture for two minutes at room temperature.
3. Add Carbonate Stop Buffer (0.20M) to the cuvette to make a total volume of 2.5 mL. Mix.
4. Take a fluorescence measurement immediately.
5. Repeat steps 3.4.1 through 3.4.4 with each standard (Table 1).

Table 1

Generate a standard curve of fluorescence versus alkaline phosphatase concentration (Figure 1).

Figure 1: Fluorescence of DiF4MU from reaction of Alkaline Phosphatase with 10 µg/mL DiFMUP and quenched with 100mM NaCO3. The fluorescence value of the reagent blank may be subtracted from that of each sample.

3.5 Alkaline Phosphatase Samples

1. Add 0.5 mL 10µg/mL DiFMUP Working Solution to each sample cuvette.
2. Add 100 µL of sample to a cuvette, invert to mix.
3. Incubate the mixture for two minutes at room temperature.
4. Add 1.9 mL Sodium Carbonate Solution to the cuvette to make a total volume of 2.5 mL. Mix.
5. Take a fluorescence measurement immediately.
6. Repeat steps 3.5.1 through 3.5.5 with each sample.
7. Calculate the amount of alkaline phosphatase from the fluorescence measurement and the linear equation determined from the AP standard concentration vs. fluorescence, step 3.4.6.

4. References

1: Brandenberger, H., and Hanson, R., Spectrophotometeric Determination of Acid and Alkaline Phosphatases, Helv. Chim. Acta, 36, 900, 1953.

2: Fernley, H. N. and Walker, P. G., Kinetic Behaviour of Calf-Intestinal Alkaline Phosphatase with 4-Methylumbelliferyl Phosphate, Biochem. J., 97, 95, 1965.