b Measurement of Transient Fluorescence Signals after Photochemical Release of ATP

The dye RH421 can be exploited also in time-resolved measurements with caged ATP to obtain kinetic information. Caged ATP is an inactive, photolabile precursor of ATP; upon exposure to an intense light flash, ATP is released and an ATP-concentration jump is creat-ed (Fig. 29). At pH 7.0, ATP is releascreat-ed from cagcreat-ed ATP with a time constant of 4.6 ms, al-lowing the detection of reactions with time constants of 10 ms or higher (147,148).

Figure 29. Photochemical release of ATP from caged ATP (P3-[1-(2-Nitrophenyl)ethyl]-ATP).

The time-resolved fluorescence signals induced by photochemical release of ATP have been measured with a home-made instrumental setup as described recently in (22) (Fig. 30).

The photochemical release of ATP is triggered by a UV flash generated by an EMG 100 excimer laser (Lambda Physics, Göttingen, Germany; wavelength 351 nm, duration 14 ns, maximum power 6 MW). A UV cut-off filter reduces the effect of the UV flash. The dye RH421 is excited by a HeNe laser set at 594 nm. A quartz lens widens the laser beam to il-luminate the whole solution almost homogeneously. The emitted light is collected by an el-lipsoidal mirror and converged onto the cathode of a photomultiplier (Mod. R928, Hamamatsu Photonics, Japan). An interference filter (663 ± 18 nm) selects the emitted light of the styryl dye before it enters the photomultiplier. The output current is amplified and digitized by a 12-bit data acquisition board of a PC with sampling frequencies between 1 and 500 kHz. The bottom of the cuvette is in contact with a thermostated copper socket that also stops the incident light.

Figure 30. Scheme of the home-made instrumental setup used for the measurements of transient fluorescence signals after photochemical release of ATP.

The measurement of the time-dependent fluorescence signal after photochemical release of ATP at saturating concentrations of Na⁺ ions and ATP enables the evaluation of the time constant of the E₁-P → P-E₂ conformational transition (128-130). At 50 mM NaCl, the enzyme is in the state Na₃E₁; ATP release triggers the reaction Na₃E₁ → Na₃E₁^.ATP → (Na3)E1-P → P-E2Na3 → P-E2 + 3 Na⁺. A fluorescence increase is recorded, corresponding to the translocation and then the release of the Na⁺ ions at the extracellular side of the membrane (Fig. 31). At saturating ATP concentrations, ATP-binding and phosphorylation are fast compared to the conformational transition. Thus, the time constant of the fluorescence increase reflects the rate of the conformational transition.

Materials

 Imidazole (Merck, buffer substance, ACS)

 EDTA (Merck, Titriplex® II, for analysis, ACS)

 MgCl2 hexahydrate (Merck, for analysis, EMSURE®, ACS)

 NaCl (Merck, for analysis)

 NPE-caged-ATP disodium salt (Molecular Probes)

 Apyrase VI (Sigma)

 RH421 (MoBiTec)

 EtOH (Merck, for spectroscopy)

Solutions

 Buffer: 25 mM imidazole, 1 mM EDTA, pH 7.2 (HCl)

 1 M MgCl₂

 200 μM RH421 in EtOH

 5 M NaCl

 10 mM cg-ATP

To remove traces of free ATP from the sample of caged ATP, Apyrase VI (1.4 x 10^-3 Units/ml) and 1.4 mM MgCl2 are added to the stock solution.

Procedure

The experiment is performed in a darkened environment at 20 ± 0.5 °C.

1- A cylindrical quartz cuvette (internal diameter 7.8 mm) is filled with 300 μl of Buffer containing 5 mM MgCl2, 200 nM RH421, 50 mM NaCl, 9 μg/ml of protein, and 100 µM caged ATP. The cuvette is equilibrated for 10 min inside the instrument to stabilize the desired temperature. During this time, it is protected from light by a cover to prevent unintended photochemical release of ATP.

2- The cuvette is uncovered and the experiment is started. The generation of a light flash by the excimer laser is controlled by the program DASYLab9, which also rec-ords the fluorescence signal. After photochemical release of ATP, the enzyme is phosphorylated and undergoes the conformational transition to P-E₂. In this confor-mation, the protein releases the Na⁺ ions on the extracellular side of the membrane.

In the absence of K⁺ ions, dephosphorylation is very slow (130) and the enzyme is trapped in the P-E2 state with virtually empty ion-binding sites since the release of Na⁺ ions is not counterbalanced by binding of protons (32). Therefore, a rapid fluo-rescence increase is detected corresponding to the electrogenic release of all three Na⁺ ions.

To allow the comparison between different experiments, the fluorescence increase is normalized with respect to the fluorescence level before ATP release. The normalized tran-sient can be fitted with a single exponential function (Eq. 3) to obtain the corresponding time constant, τ.

Equation 3 F_normF_max (1e^t) with

Fnorm = normalized fluorescence

F_max = normalized fluorescence level of the steady-state after ATP release τ = time constant of the normalized fluorescence increase after ATP release

Figure 31. Transient fluorescence signal recorded after photochemical release of ATP at saturating concentrations of Na⁺ ions and ATP. The signal can be fitted with a single exponential function.

At non-saturating ATP concentrations, ATP-binding becomes rate-limiting and the time constant of the fluorescence increase reflects the rate of the ATP-binding reaction. Since only part of the enzyme in the cuvette is phosphorylated and undergoes the conformational transition, the amplitude of the signal is lower than at saturating ATP concentrations. Fitting of the time constant or of the amplitude of the normalized fluorescence increase versus the ATP concentration with the Michaelis-Menten function (Eq. 4) provides the apparent ATP-binding affinity (130).

Equation 4

 





K

ATP

M 

 

 ( _{min max})

max



 

 with

τ = time constant of the normalized fluorescence increase after ATP release τmax = time constant at the lowest ATP concentration

τmin = time constant at the highest ATP concentration [ATP] = concentration of ATP

K_M = half-saturating ATP concentration

To obtain a reliable value of ATP-binding affinity, the concentration of free ATP re-leased by caged ATP under the experimental conditions has been determined using the luciferin/luciferase test (149). About 10% of ATP is released from caged ATP by a single flash.

The reduction of the number of data points obtained by the program DASYLab9 and the normalization of each fluorescence trace have been performed with the program Redulite2.

The signal output has been processed with the program Drifter and the fitting procedure has been performed with the data elaboration program FigP 2.98. The error is expressed as SEM.