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Reentrant Phase Behavior in Protein Solutions Induced by Multivalent Salts: Strong Effect of Anions Cl - Versus NO 3

Reentrant Phase Behavior in Protein Solutions Induced by Multivalent Salts: Strong Effect of Anions Cl - Versus NO 3

Journal of Physical Chemistry. B 122(50): 11978-11985

In this work, the effects of the two anions Cl- and NO3- on the phase behavior of bovine serum albumin (BSA) in solution with trivalent salts are compared systematically. In the presence of trivalent metal salts, negatively charged proteins such as BSA in solution undergo a reentrant condensation (RC) phase behavior, which has been established for several proteins with chlorides of trivalent salts. Here, we show that replacing Cl- by NO3- leads to a marked change in the phase behavior. The effect is investigated for the two different cations Y3+ and La3+. The salts are thus YCl3, Y(NO3)3, LaCl3, and La(NO3)3. The experimental phase behavior shows that while the chloride salts induce both liquid-liquid phase separation (LLPS) and RC, the nitrate salts also induce LLPS, but RC becomes partial with La(NO3)3 and disappears with Y(NO3)3. The observed phase behavior is rationalized by effective protein-protein interactions which are characterized using small-angle X-ray scattering. The results based on the reduced second virial coefficients B2/ B2HS and 1/ I( q → 0) demonstrate that the NO3- salts induce a stronger attraction than the Cl- salts. Overall, the effective attraction, the width of the condensed regime in the RC phase diagram, and the nature of LLPS follow the order LaCl3 < YCl3 < La(NO3)3 < Y(NO3)3. Despite the decisive role of cations in RC phase behavior, isothermal titration calorimetry measurements indicate that replacing anions does not significantly influence the cation binding to proteins. The experimental results observed are discussed based on an "enhanced Hofmeister effect" including electrostatic and hydrophobic interactions between protein-cation complexes.

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Accession: 065900699

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PMID: 30461282

DOI: 10.1021/acs.jpcb.8b10268

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