Solubility of recombinant human tissue factor pathway inhibitor

Study of recombinant human tissue factor pathway inhibitor (rhTFPI) solubility shows (1) an inverted bell-shaped pH-solubility profile with a broad solubility minimum between pH 5 and 10 such that the solubility minimum midpoint is 2-3 pH units away from its isoelectric point; (2) a negative temperature-solubility coefficient; (3) a strong dependence of solubility on the valence of electrolytes, with both multivalent cations and anions enhancing this effect; and (4) a significant increase of solubility in the presence of charged polymers. At pH 6-7, rhTFPI solubility-salt profiles display typical salting-in and salting-out biphasic effects. At a slightly lower pH (pH 5), a third phase in addition to the salting-in and salting-out phases was observed at low ionic strength conditions (5 to 50 mM) where rhTFPI solubility increased as salt concentration decreased. The salting-out constant for rhTFPI in NaCl is 1.04 M(-1) and is independent of the pH of the solution. Resolubilization of rhTFPI precipitates revealed that "insolubility precipitates" (seen during buffer exchanges) resulted from protein solute saturation and could be redissolved by "native" solvent conditions. On the other hand, "instability precipitates" (typically seen after exposure to elevated temperatures or extended storage periods) were caused by insoluble protein aggregate formation and required strongly denaturing conditions to redissolve.