Determination of the Water Sorption by Protein Immersed in Water Organic Solvent

1. Place 4-10 mg of solid protein preparation and 4.0 mL of organic solvent containing a certain fraction of water in a preweighed thin-bottom glass ampoule and close this ampoule with a preweighed cap (see Note 11). The solid:liquid ratio is chosen close to the value used in the calorimetric experiment.

2. Maintain the ampoule at a constant temperature (25°C) during some time. This time period is not less than the time corresponding to the completion of the heat evolution in a given solvent as found by calorimetry (see Subheading 3.1.2. and Note 8).

3. Remove the aliquot of the solvent from the ampoule with a syringe. The volume of aliquot varied from 0.1-0.3 mL at high water contents in the solvent to 1-1.5 mL at low water contents.

4. Weigh the syringe filled with a solvent aliquot, and then transfer the solvent into the Fischer reagent medium. Then, the total water amount introduced by the aliquot should be measured electrochemically.

5. Weigh the syringe again and obtain the aliquot weight by the difference. The water concentration in the solvent should be calculated from the measured water amount in the aliquot, the weight of aliquot, and the solvent density.

6. Repeat such extraction of the aliquots and the electrochemical measurement two to three times for 40-60 min. Reproducible values of water content in the solvent indicate the attainment of equilibrium (see Note 12).

Fig.3. AH values (per gram; filled diamonds) and water sorption by HSA (open circles) plotted against water concentration in acetonitrile (298 K). The dashed line shows the initial water amount on the HSA (10%, w/w). (After ref. 11.)
Fig.4. AH values (per gram; triangles) and water sorption by HSA (circles) plotted against water concentration in 1,4-dioxane (298 K). The dashed line shows the initial water amount on the HSA (10%, w/w). (After ref. 13.)

7. Withdraw the bulk of the liquid phase from the ampoule with a syringe and weigh the ampoule again. The apparent weight of the remaining liquid phase is calculated as the difference between the final weight of the sealed ampoule and the sum of the weights of the empty ampoule, the cap, and the dry protein.

0 2 4 6 8 10 Water concentration, mol/L

Fig.5. AH values (per gram) plotted against water concentration in pyridine (298 K). (After ref. 13.)

8. Put this ampoule containing the protein and a small amount of the remaining water-organic mixture into the Fischer apparatus.

9. Break off the bottom of the ampoule and measure the total amount of water on the protein sample (and in the remaining liquid).

10. Weigh the cap after the experiment in order to determine the amount of the solvent that could have been adsorbed by the cap.

11. Subtract the difference in the weight of the cap from the apparent weight of the remaining solvent. The final result is the true weight of the remaining liquid phase.

12. Calculate the amount of water in the remaining liquid using the weight of the remaining liquid phase, its water content measured previously, and the liquid density.

13. The amount of water on the HSA corresponds to the difference between the total measured amount of water in the ampoule and the amount of water in the remaining liquid phase. This bound amount of water is expressed as percentage by weight with respect to the dry protein (%, w/w) (see Note 13).

Water sorption isotherms are exemplified in Figs. 3 and 4 (see Note 14). As is seen, the complicated shape for calorimetric data in 1,4-dioxane is supported by a similar pattern for the water amount bound to HSA. The smooth sorption isotherm in acetonitrile mixtures follows the pattern for calorimetric data. Additional examples combining sorption and calorimetric data for HSA preparation in different solvents are presented in refs. 12 and 14.

0 0

Post a comment