Proteins are biological macromolecules build of amino acids. They differ in number and
sequence of amino acids that are linked together (primary structure). This affects protein
coagulation in its native form (secondary, tertiary and quaternary structure). Proteins have
many different roles in living organism: structural, functional and regulatory. The
stability of a given protein in solution is affected by its concentration, pH value and type
of buffer, type and concentration of additives (salt, sugars, polymers), temperature,
pressure etc. The study of the conditions of phase stability of proteins in solutions
(denaturation, aggregation, phase separation) is one of the key factors in the development
of drugs, as the effectiveness and shelf life of a biological drug depend on it. Protein
aggregation results in a number of disease states (Alzheimer's, Parkinson's, cataracts),
while phase separation can also be a desirable phenomenon, for example in protein
purification (crystallization).
As part of my master's thesis, I studied the effect of polyethylene glycol (PEG) and salt
on the phase stability of bovine serum albumin (BSA) in acetate buffer at two pH values
(near and below the isoionic point). I used PEG with molar masses of 6000, 10000 and
20000 g/mol and various sodium salts (NaF, NaCl, NaBr, NaI, NaNO3, NaOAc) and
alkali chlorides (LiCl, NaCl, KCl, RbCl, CsCl). The BSA concentration was 90 mg/mL
in all cases. The solutions were prepared in 0.1 M acetate buffer with pH = 4.6 and 4.0.
PEG destabilizes the solution (shifts the cloud point temperature to higher values) the
higher its molar mass and the higher its concentration. The effect of added PEG is
observed earlier in solutions with pH = 4.6 than in 4.0. With increasing salt concentration,
the effect of salt is stabilizing at pH = 4.6 and destabilizing at pH = 4.0. Ionospecific
effects are seen in both anions and cations. There is a correlation between Tc and the
standard free enthalpy of hydration of anions / cations of the added salt. At pH ≈ pI, an
ion with a more negative standard free hydration enthalpy is considered to increase Tc
(destabilize the solution), while at pH < pI, such ion is considered to decrease Tc (stabilize
the solution).
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