3.2.1. Calorimetric Titration Study (see Notes 6-7)
1. Load the immobilized protein (0.25 g) into the calorimeter titration cell and add 1 mL of a suitable buffer (for example acetate buffer 50 mM, pH 5.0).
2. Prepare 10 mL of 3'-CMP inhibitor solution 1 mMin the same buffer (10 immoles).
3. Pump the inhibitor solution at a constant rate (ranging from 0.07 to 0.3 mL/min) until the heat effect decreases to baseline values (about 20 min).
4. Prepare a second sample with the same amount of supporting material without enzyme and add the inhibitor with the same rate. This experiment is a control run. The aim is to obtain a suitable baseline. The heat effects in these conditions in the absence of enzymes are minimal.
3.2.2. Calorimetry (see Notes 2-5)
Sensitive isothermal batch calorimeters are commercially available. The results presented in Table 2 were obtained with a Titration Activity Monitor (TAM, Ther-mometric, Jarfalla AB, Sweden), equipped with twin titration-cells. The VP-ITC (Microcal), a heat compensation and titration calorimeter, is a suitable instrument as well (vessel volume 1.3 mL, detection limit 0.8 ^W).
Isothermal batch calorimeters are the method of choice for studying reaction with immobilized proteins. Flow instruments cannot be used because heterogeneous materials cannot be introduced accurately inside the calorimeter. Instead, batch calorimeters can quantitatively accommodate the material to be titrated inside the instrument cell and are readily accessible to an external flowing reagent. However, batch instruments require a relatively long time for thermal equilibration and this may be a serious drawback if several samples have to be tested. On the other hand, batch calorimeters have the sensitivity to examine slow reactions and the potentiality to analyse a full protein-ligand titration curve with a single run.
Analysis of the data was implemented by standard theory and software (19,20). The theory of the Tian correction is well documented from theoretical and experimental point of view (21,22). Algorithms for the calculation of the Tian effect may be available commercially (for instance, the algorithm developed by Kirchoff, NBS, Gaithersburg, MD).
3.3. Adsorption: Thermal Unfolding
3.3.1. Protein Immobilization and Hydration
1. Dissolve 30 mg of RNase in 5 mL of phosphate buffer.
2. Add the protein solution to 1 g of celite and mix throughout to form a homogeneous slurry. Spread the paste on a wide flat glass surface of suitable dimensions (10-cm diameter or more).
3. Prepare a mixture of water and H2SO4 at a fixed volume ratio. The volume ratio may varies from 5 to 20%, according to standard reference tables (26). The higher the sulfuric acid concentration the lower the final water content. Twenty milliliters of solution are sufficient for the isopiestic equilibration. Use a wide open glass container such as a glass Petri dish.
4. Transfer the protein-celite slurry and the sulfuric acid-water solution into a vacuum-tight dry desiccator.
5. Allow the isopiestic equilibration to take place for a week at constant temperature (for instance at 25 ± 1°C; see Note 8). By changing the water/H2SO4 ratio, the total water content of the protein sample may be varied from 5 to 30% (w/w) corresponding to 0.24 to 1.4 g/g of protein.
6. For very low hydration level (<0.2 g/g) the sample can be dried under high vacuum for different time intervals.
7. Measure the final water content of the protein sample by standard Karl Fisher water titration (see Notes 9 and 10).
8. Check the concentration of RNase in the dried sample by suspending the celite powder by weight in a known amount of aqueous buffer solution (phosphate buffer 100 mM, pH 7.0). Mix the sample vigorously and centrifuge at 4000-5000 rpm for 1 min in an Eppendorf centrifuge 5415 or 5417. Read the absorbance at 278 nm of the supernatant (e278 = 9700 mol/cm). Many proteins, including RNase, are completely released from celite in an aqueous solvent.
3.3.2. Calorimeter Experiments (see Notes 12 and 13)
1. Protein samples were directly withdrawn from the desiccator and added to the pan. This did not alter significantly the hydration level of the sample providing that only few minutes elapsed from sample withdrawal to pan sealing.
2. Load a calorimeter pan with 20-25 mg of sample (containing 2-3 mg of protein), which are sufficient to observe a good signal-to-noise ratio.
3. When needed, add 20-30 L of solvent, either aqueous or organic (anhydrous).
4. Allow the samples to equilibrate for 30 min before starting the thermal ramp. The same amount of inert support and solvent was used in the reference pan in order to obtain an automatic baseline compensation.
5. Use an appropriate scan rate. Usually a scan rate range from 1 to 10°C/h are suitable (see Note 11).
3.3.3. Calorimetry (see Notes 2-5)
The best instruments to study the thermodynamic properties of adsorbed proteins are those batch calorimeters with sealed pans as sample holders. Because the amount of the sample, usually powdered material containing the adsorbed enzyme, has to be known precisely, the most convenient method of preparation, therefore, is to weigh the sample (and solvent) directly on the pan. Otherwise, precise quantitative cell loading may be difficult with a standard calorimetric cell usually built with small entrance holes.
Another important factor is the temperature range of the instrument. The calorimeters mentioned in Subheading 2. were developed for studies using water as a solvent and cannot span wide temperature ranges. Conversely, batch calorimeters using pan sample holders are devoted to studying temperature effects up to several hundreds of degrees centrigrade. For instance, the results of the DSC experiments showed in Table 3 were obtained with the Perkin Elmer DSC-7 calorimeter (Norwalk, CT), which is able to span from -170°C up to 700°C.
The Mettler Toledo DSC 822 calorimeter (Columbus, OH) is a more recent model. The standard aluminum pans contain only 10-15 pL of sample. Nevertheless, this calorimeter is a sensitive instrument, reaching a nominal resolution value <0.4 pW. Crucible type pans can be used, which can contain up to 125 pL of sample and can hold 20 bars of internal pressure.
The best fitting and deconvolution analysis were carried out according to the two-state approximation mathematical model described in the literature, as already mentioned in Subheadings 1.2. and 3.1.5.
1. Controlled pore glass beads can be purchased in different size and pore diameter (from 700 to 1400 A) from Fluka. They are available as neat, unmodified or chemically modified glass. There are several type of chemical derivatives, including the aminopropyl group. The density of the functional group varies with the preparation. With the aminopropyl beads derivative, pore size 500 A, 30-45 mesh, the amount of modified aminopropyl groups can be as a high as 2 mmol/g
2. Special care has to be taken on degassing because of the heterogeneity of the samples. Careful treatment under reduced pressure or the use of extensively degassed buffer solutions is required in order to avoid the formation of air bubbles during the thermal scan.
3. Another practical difficulty with all the instruments is the determination of the exact amount of material (immobilized protein) actually used to fill the calorimeter cell. The difficulty may arise from the dimensions of the calorimetric sample cell entrance hole, which is often quite small. The best approach is to standardize the quantity before adding the material to the cell. For instance, a small glass container (a small beeker or, better, a small funnel) may be used, whose volume is calibrated. A known amount of solid support (containing the protein) may then be routinely obtained by filling the container with a fixed amount of material. This can be easily measured by weight or volume (by referring to calibration marks on the container). Then the whole amount is poured into the calorimetric cell. The amount of material for each run ranges from 0.05 to 0.2 g of support, containing about 1.5-6.0 mg of protein.
4. Thermal scan rates may vary from 30 to 120°C/h, as in the case of the free enzymes in solution. Data collection every 10-15 s usually suffices.
5. Reference scans of sample containing only the supporting material (without proteins) are necessary, although many materials (as the glass beads) do not show any significant temperature-dependent heat effects (at least up to 100°C).
6. During the titration experiment, in order to minimize the eventual grinding of the material, switch on the stirring of the sample immediately before starting the external pump.
7. The stirring of the heterogeneous sample within the calorimetric cell is another important issue. According to our experience this is a critical parameter. It is necessary to ensure a complete and thorough mixing of the reagent, otherwise spurious results may be obtained. The stirring device offered by the manufacturer may not be fully suitable and sometimes optimization is needed. For instance, the blades of the mechanical stirrer have the highest efficiency if tilted in such a way to push the sediment in an upward motion, from bottom to top.
8. During isopiestic equilibration, at very high concentration of sulfuric acid (water content <5%) it is possible that acidic components may evaporate in the vapor phase, depending upon the temperature. The presence of volatile acidic compounds can be detected by pH indicator paper added inside the dessicator. In this case it is necessary to pay attention to the possible alteration of the protein sample.
9. Most of the water present in the sample after isopiestic equilibration is used up to hydrate the protein molecules. In fact, control experiments showed that celite itself retains only a very small amount of water when equilibrated in the same conditions without protein.
10. When working with volatile solvents it is necessary to use high-pressure pans during the calorimeter run. These pans are usually provided by the manufacturer as accessories. The experiments with RNase were performed with capped stainless steel pans with an internal volume of about 80 |L, resistant to inner pressures as high as 25 bars.
11. In the case of RNase, which has a well characterized and reversible unfolding transition, the scanning rate was as high as 10°C/min. The thermodynamic parameters of unfolding were independent of the scanning rate from 5 to 15°C/min.
12. Note that, although the Perkin Elmer DSC-7 calorimeter is rather an old model, it functions on a true calorimetric principle (i.e., the power compensation "null-balance" principle). This means that the energy absorbed or evolved by the sample is compensated by addition or subtraction of an amount of electrical energy equivalent to the sample pan holder with respect to the reference pan holder. The energy and not a temperature difference is the direct output of the instrument.
13. The Mettler Toledo DSC 822 calorimeter (Columbus, OH) is a recent model. Unlike Perkin Elmer calorimeters, this instrument is based on the principle of measuring the temperature difference between sample and reference cells. Nevertheless, the sensitivity is as high as 0.4 |W.
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