CARACTERIZACION DE LA ZONA 1-2-3 COMUNIDAD CAMPESINA

When using visible laser pulses for inducing temperature jumps, heat transducers are needed to absorb the laser pulse energy[101]. Typically, triphenylmethane dyes, such as basic fuchsin (BF) and crystal violet (CV) are used for such purposes because they are known to undergo rapid internal conversion on the sub-ps timescale[95, 97, 102, 103]. Both dyes have a high extinction coefficient at 532 nm and have been used in our research group to obtain successful temperature jumps.

The molecular structure of CV is shown in Figure 3.1. It is the first dye to be tested as heat transducer and was successfully used to achieve temperature jumps in protein and peptide samples[19, 101, 104]. However, this dye is not the best heat transducer since it bleaches rapidly upon exposure to laser light, thus preventing the accumulation of several single scans without moving or flowing the sample. To circumvent this problem, the sample was flowed using a peristaltic pump to continuously exchange the dye

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molecules so as to minimize the bleaching effect. However, flowing the sample is disadvantageous since it requires a large amount of protein/peptide sample, which is not always available, and most importantly, flowing the sample leads to a massive increase in cavitation events. This is because flowing the sample increases the probability of finding dye aggregates in the heated volume which act as nucleation sites for these cavitation events (see chapter 2.4.2).

Figure 3. 1: Molecular structure of crystal violet

Other dyes were later tested[92] and BF was found to be a suitable dye for temperature jump measurements. As shown in Figure 3.2 below, the chemical structure of BF is very similar to the chemical structure of CV with the exception of the groups attached to the phenyl rings. The internal conversion is even faster than that of CV because of the smaller attached groups[102].

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Pozo Ramajo[92] found that the BF dye bleaches considerably slower than CV and that it seemed possible to accumulate 512 single measurements when the sample was not being flowed. However, when looking more carefully, we found that it was not possible to accumulate 512 single measurements since the dye begins to bleach after 15 seconds of irradiation (~150 single measurements). Furthermore, even when flowing the sample every 15 seconds, the temperature jump decreased by 5% over 5 minutes, accompanied by accelerated dye bleaching and increased cavitation. Thus, our aim here was to develop a technique to minimize these artifacts.

The need to flow the sample regularly requires the use of a small reservoir. Initially, the dye solution was stored in a plastic syringe and the absorbance was monitored over a twenty-four hour period (data not shown). We found that the dye solution degrades rapidly within three hours after preparation. This is because the dye adsorbs to the plastic materials, thus decreasing the concentration of the solution. We then investigated several materials and found that a glass reservoir connected to the cell through Teflon tubing and a metal connector (see Figure 3.3) is ideal for minimizing dye adsorption. Care was taken not to allow the sample to remain in the syringe at any time to avoid dye adsorption on the plastic material.

Figure 3. 3: Schematic of the temperature controlled cell with CaF2 windows (50 µm spacing) equipped with glass reservoir, syringe and Teflon tubing.

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The temperature-controlled cell was fitted with two CaF2 windows and a cut 50

µm spacer. The syringe was filled with ~0.8 mL BF dye solution and attached to the temperature controlled IR cell. The sample was then pumped through the cell with the syringe ensuring that no bubble formation occurs. Since the dye adsorbed to the plastic syringe, it was ensured that the solution did not remain in the syringe for any length of time by pumping the solution into the glass reservoir (see schematic above). Finally, the sample cell was placed on a mount that was fitted with micrometer screws (used for moving the cell) and placed at the sample position (see Figure 2.1).

For temperature jump measurements, the sample was exchanged every 12-15 seconds using the syringe as a pump and the cell was moved after 150 seconds to use a fresh sample spot. This was to ensure that dye aggregates would not amass on the cell window which leads to an increase in cavitation events. This was proven to be an effective method and was useful for taking decent temperature jump measurements, as it allowed us to maintain a constant temperature jump over the full acquisition process. Therefore, this newly hand-operated design allowed us to reduce the sample volume, to reduce dye photo-bleaching and dye adsorption, and to maintain a constant temperature jump.

We also found that while trying to obtain the highest temperature jump possible, the dye degrades rapidly when the energy of the laser is above 2 mJ. To maintain a constant temperature jump throughout, the laser energy was kept as low as possible (~1 mJ), yielding a temperature jump of 3 oC.

For the protein investigated in chapter 4 (WT-BBL), a high salt concentration (200 mM NaCl) is needed as this has been proven to stabilize the native state of the protein. It is essential that temperature jump measurements are taken in conditions suitable for the protein’s stability and comparable to previous studies of this protein.

Since heat transducing dye is needed for the induction of temperature jumps in a protein/peptide sample, it is necessary to know how the dye is influenced by the addition of NaCl. Here we tested whether BF dye was stable in the presence of high salt concentration.

Figure 3.4 shows the absorption spectra of BF/D2O solution at various salt

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similar in shape in the presence and absence of NaCl. It is important to mention that the dye solution was prepared at a higher concentration (~5 x 10-3 M), greater than the concentration typically used for measurements (~5 x 10-4 M). However, the dye precipitated upon increasing NaCl concentration. For example, the saturation dye concentration at 50 mM NaCl is ~6 x 10-4 M and the saturation dye concentration at 200 mM NaCl is ~2 x 10-4 M, as determined from the spectra in Fig. 3.4.

Figure 3. 4: Absorbance spectra of a solution of Basic Fuchsin in D2O (~5 mM) with various concentrations of NaCl, ranging from 0 to 200 mM, 50 µm spacing.

For the solution containing 50 mM NaCl, we were able to induce temperature jumps of ~4 oC. This temperature jump was possible since the dye solution gave an absorbance of ~0.2 O.D. (typical absorbance for inducing temperature jumps). However, above 50 mM NaCl concentration, the saturation concentration of the dye is too small to get sufficient temperature jumps.

Even though we were able to perform decent temperature jumps in a 50 mM NaCl concentration solution, this is not ideal for the protein investigated (see chapter 4), because changing the ionic strength would require changing the protein’s preparation protocol and it would not allow for comparability to previous results. For this reason, BF dye cannot be used for inducing temperature jump in the protein BBL samples.

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Therefore, other heat transducers are needed for the investigation of the protein WT- BBL.