Solubility of recombinant human tissue factor pathway inhibitor

Study of recombinant human tissue factor pathway inhibitor (rhTFPI) solubil ity shows (1) an inverted bell-shaped pH-solubility profile with a broad so lubility minimum between pH 5 and 10 such that the solubility minimum midpo int is 2-3 pH units away from its isoelectric point; (2) a negative tempera ture-solubility coefficient; (3) a strong dependence of solubility on the v alence of electrolytes, with both multivalent cations and anions enhancing this effect; and (4) a significant increase of solubility in the presence o f charged polymers. At pH 6-7, rhTFPI solubility-salt profiles display typi cal 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 solubi lity increased as salt concentration decreased. The salting-out constant fo r rhTFPI in NaCl is 1.04 M-1 and is independent of the pH of the solution. Resolubilization of rhTFPI precipitates revealed that "insolubitity precipi tates" (seen during buffer exchanges) resulted from protein solute saturati on and could be redissolved by "native" solvent conditions. On the other ha nd, "instability precipitates" (typically seen after exposure to elevated t emperatures or extended storage periods) were caused by insoluble protein a ggregate formation and required strongly denaturing conditions to redissolv e.