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Izmed vseh molekul, ki jih najdemo v živih organizmih, so proteini najbolj zastopani in s funkcionalnega stališča najbolj raznoliki. Sestavljeni so iz dolgih verig aminokislin in na njihovo konformacijo, strukturo ter stabilnost lahko vplivajo številni parametri. Vodne raztopine proteinov običajno vsebujejo soli, najbolj pogosto v obliki pufra. Poleg proteinov, soli in vode pa so v bioloških sistemih pogosto prisotne še druge makromolekule (lipidi, sladkorji, nukleinske kisline), in to v znatnih koncentracijah. Vpliv zasedenosti prostora s temi makromolekulami lahko v laboratorijskih razmerah ponazorimo z dodatkom nevtralnega polimera polietilen glikola. Soli in druge molekule tudi vplivajo na interakcije molekul proteina z raztopino. Eden izmed načinov ugotavljanja teh interakcij je merjenje zeta potenciala. To delo zajema meritve zeta potenciala v raztopinah proteina lizocima, kjer je bil prisoten tudi polietilenglikol v različnih koncentracijah (0, 100 in 150 g/l) in pri treh vrednostih pH (4,1, 7 in 9). Vedno je bil prisoten enostaven elektrolit (10 mM NaBr). Viskoznost raztopin polietilenglikola narašča z njegovo koncentracijo in tudi z dolžino polimerne verige. Z naraščajočo koncentracijo pa se zmanjšuje dielektrična konstanta raztopine. Rezultati kažejo, da zeta potencial narašča s koncentracijo polietilenglikola pri vseh treh izbranih pH vrednostih. Zeta potencial narašča tudi z dolžino polimerne verige. Poleg lizocima sem uporabila tudi protein goveji serumski albumin. Tudi pri tem proteinu se zeta potencial z naraščajočo koncentracijo polietilenglikola po absolutni vrednosti povečuje. Pri pH 7 je namreč naboj BSA negativen. Rezultati meritev zeta potenciala v sistemih brez polietilenglikola so primerljivi s tistimi v literaturi. Meritve v prisotnosti polietilenglikola pa še niso bile narejene. Proteins are the most abundant molecules found in living organisms, and, from a functional point of view, also the most diverse. They consist of long chains of amino acids and their conformation, structure and stability can be influenced by many parameters. Aqueous solutions of proteins usually contain salts, most often in the form of a buffer. In addition to proteins, salt, and water, other macromolecules (lipids, sugars, nucleic acids) are often present in biological systems. In laboratory conditions, influence of these macromolecules can be mimicked by the addition of the neutral polymer polyethylene glycol. Salts and other molecules affect the interaction between protein molecules. One way to determine these interactions is by measuring the zeta potential. In this work, I measured the zeta potential in lysozyme protein solutions, where polyethylene glycol was also present in different concentrations (0, 100 and 150 g/L) and at three pH values (4,1, 7 in 9). 10 mM NaBr salt was used in all cases. The viscosity of polyethylene glycol solutions increases with its concentration and also with the length of the polymer chain. As the concentration increases, the dielectric constant of the solution decreases. The results show that the zeta potential increases with polyethylene glycol concentration at all three pH values. The zeta potencial also increases with the length of the polymer chain. In addition to lysozyme, I also used the protein bovine serum albumin. For this protein, the zeta potencial increases in absolute value with increasing polyethylene glycol concentration. At pH 7, the charge of bovine serum albumin is negative. The results of zeta potential measurements in systems without polyethylene glycol are comparable to those in the literature. However, measurements in the presence of polyethylene glycol have not yet been made.

The stability of bovine serum albumin (BSA) solutions against phase separation caused by cooling the system is studied under the combined influence of added poly(ethylene glycol) (PEG) and alkali halide salts in water as solvent. The phase stability of the system depends on the concentration of the added PEG and its molecular mass, the concentration of the low molecular mass electrolyte and its nature, as also on the pH of the solution. More specifically, the addition of NaCl to the BSA-PEG mixture promotes phase separation at pH = 4.0, where BSA carries the net positive charge in aqueous solution, and it increases the stability of the solution at pH = 4.6, i.e., near the isoionic point of the protein. Moreover, at pH = 4.6, the cloud-point temperature decreases in the order from NaF to NaI and from LiCl to CsCl. The order of the salts at pH = 4.0 is exactly reversed: LiCl and NaF show the weakest effect on the cloud-point temperature and the strongest decrease in stability is caused by RbCl and NaNO$_3$. An attempt is made to correlate these observations with the free energies of hydration of the added salt ions and with the effect of adsorption of salt ions on the protein surface on the protein–protein interactions. Kosmotropic salt ions decrease the phase stability of BSA-PEG-salt solutions at pH < pI, while exactly the opposite is true at pH = pI.

Pharmaceutical design of protein formulations aims at maximum efficiency (protein concentration) and minimum viscosity. Therefore, it is important to know the nature of protein-protein interactions and their influence on viscosity. In this work, we investigated the dependence of the viscosity of BSA in an aqueous 20 mM acetate buffer at pH = 4.3 on protein concentration and on temperature (5–45 °C). The viscosity of the solution increased with protein concentration and was 230% higher than the viscosity of the protein-free formulation at 160 mg/mL. The viscosity decreased by almost 60% in the temperature range from 5 to 45 °C. The agreement of the modified Arrhenius theory with experiment was quantitative, whereas a hard-sphere model provided only a qualitative description of the experimental results. We also investigated the viscosity of a 100 mg/mL BSA solution as a function of the concentration of added low molecular weight salts (LiCl, NaCl, KCl, RbCl, CsCl, NaBr, NaI) in the range of salt concentrations up to 1.75 mol/L. In addition, the particle size and zeta potential of BSA-salt mixtures were determined for solutions containing 0.5 mol/L salt. The trends with respect to the different anions followed a direct Hofmeister series (Cl− > Br− > I−), whereas for cations in the case of viscosity the indirect Hofmeister series was observed (Li+ > Na+ > K+ > Rb+ > Cs+), but the values of particle sizes and zeta potential did not show cation-specific effects. Since the protein is positively charged at pH = 4.3, anions are more attracted to the protein surface and shield its charge, while the interaction with cations is less pronounced. We hypothesize that salt surface charge shielding reduces protein colloidal stability and promotes protein aggregate formation.