Cuestionario a clientes de tarifa mixta y resultados del estudio empírico

Since the T-mixer allows the effective mixing of two solutions of reactants, the custom-made flow cell attached with the T-mixer was first used to detect the fast kinetics. The detection of a reaction process at different times was made by changing the length of the decay line from T-mixer to the detection flow cell. In order to record different dead time points, the solution left in the flow cell as well as the T-mixer was removed. When two solutions were ready to flow into the T-mixer, the solution injection and the data collection were started simultaneously. The time when we saw the first LD signal after a flat baseline of approximately zero, was the dead time of the system.

DNA exonuclease kinetics

An assay was then required where the signal produced is an LD signal that changes due to a change in alignment during the reaction to test the continuous channel flow LD system as a method to monitor fast kinetics. One such reaction is the enzymatic hydrolysis of DNA, a reaction in which DNA is degraded by DNase I to small fragments that have a greatly reduced or no LD signal (N’soukpoé-Kossi, Diamantoglou and Tajmir-Riahia 2008).

250 μM of ct-DNA and 0.73 μM of DNase I in the reaction buffer (50 mM Tris-HCl, pH 8.0, 1 mM Ca2Cl, 1 mM MgSO4) were stored separately in two

syringes. Nine different lengths of decay line were used in this experiment, which monitored the reaction of DNA digestion from 1.9 s to 4.4 s. 300μl of each solution was injected simultaneously at 1.5 ml/min for each decay line. Continuous channel

measured at 260 nm. Then the LD of the steady state phase was plotted against different reaction times to give the results below.

Figure 7.5.2.1 Continuous channel flow-LD system based on the custom-made flow cell was

used to measure the rapid digestion of ct-DNA by DNase I. 250 μM of ct-DNA and 0.73 μM of DNase I was stored in two syringes separately. 300 μl of each solution was injected simultaneously at 1.5 ml/min for each decay line. LD was measured at 260 nm, and plotted against different reaction time.

As seen in figure 7.5.2.1, DNA shows a negative LD signal, as discussed in the previous section (§ 7.5.1). It is clear that the intensity of LD decreases signifiantly (from 0.0017 to 0.0011 units) over the 2.5 s of DNA digestion. Thus upon being mixed with DNase I, the LD signal of DNA at 260 nm diminishes indicating that the alignment of the DNA in the flow cell is reduced. This agrees with the expectation that the action of DNase I leads to the generation of progressively shorter DNA fragments which are less able to align and hence produce a less intense LD signal.

‐0.0024 ‐0.0022 ‐0.002 ‐0.0018 ‐0.0016 ‐0.0014 ‐0.0012 ‐0.001 ‐0.0008 0 1 2 3 4 5 LD 260   (absorbance   units) Time(s) DNA + Dnase I

FtsZ assembly kinetics

Finally, the continuous channel flow-LD system based on the custom-made flow cell was used to study the early stages of FtsZ polymerization. 22 μM of FtsZ in the polymerization buffer (100 mM MES-KOH, pH 6.5, 20 mM MgCl2, and 100 mM

KCl) and 0.4 mM of GTP were stored separately in two syringes. 300 μl of each solution was injected simultaneously at 1.5 ml/min. Continuous channel flow LD was measured at 210 nm, which is the backbone region of FtsZ. The detection for the process of FtsZ polymerization at different time was made by changing the length of the decay line from T-mixer to the detection flow cell. Then the LD of the steady state phase subtracted by the value when the flow stopped was plotted against different reaction time, which is shown in figure 7.5.2.2.

Ten different lengths of decay line were used in this experiment. The FtsZ polymerization was measured from 0.6 s to 4.1 s. FtsZ polymers show positive LD, because the protein backbone axis is parallel to the light beam under the continuous flow (figure 7.5), and theirπ-π*transitions are parallel to the flow direction.

Negative control experiments were also performed. When FtsZ was injected into the flow cell alone, there no LD signal was observed, indicating that only FtsZ polymers result in the positive LD signal at 210 nm.

It can be observed from figure 7.5.2.2 that the LD of FtsZ increased slightly (from 0 to 0.001 units) over the 4.1 s of the reaction, suggesting that FtsZ protofilaments were formed gradually. However, at 0.6 s, the earliest data point monitored, there was not much increase in LD, which is consistent with the previously reported lag phase (Chen et al. 2005). This lag lasted for 1 s before a significant increase in LD was observed. The minimal kinetic model of FtsZ polymerization outlined in the literature postulates three steps after addition of GTP to FtsZ. The first step in the assembly mechanism is activation of the FtsZ monomer, which is needed to account for the lag of nearly 1 s, followed by formation of a dimer nucleus and elongation

(Chen et al. 2005). The continuous channel flow shows us the early stages of FtsZ assembly. After that, there is a plateau and decrease in FtsZ polymerization which is already shown by either light scattering or Couette flow LD data.

Figure 7.5.2.2 Continuous channel flow-LD system based on the custom-made flow cell was

used to measure the polymerization of FtsZ. 22 μM of FtsZ in the polymerization buffer (100 mM MES-KOH, pH 6.5, 20 mM MgCl2, 100 mM KCl) and 0.4 mM of GTP was stored in two

syringes separately. 300 μl of each solution was injected simultaneously at 1.5 ml/min for each decay line. LD was measured at 210 nm, and plotted against different reaction time.