In the protein purification, drug delivery, food industry, and biotechnological applications involving protein-polyelectrolyte complexation, proper selection of co-solutes and solution conditions plays a crucial role. The onset of (bio)macromolecular complexation occurs even on the so-called "wrong side" of the protein isoionic point where both the protein and the polyelectrolyte are net like-charged. To gain mechanistic insights into the modulatory role of salts (NaCl, NaBr, and NaI) and sugars (sucrose and sucralose) in protein-polyelectrolyte complexation under such conditions, interaction between bovine serum albumin (BSA) and sodium polystyrene sulfonate (NaPSS) at pH = 8.0 was studied by a combination of isothermal titration calorimetry, fluorescence spectroscopy, circular dichroism, and thermodynamic modeling. The BSA-NaPSS complexation proceeds by two binding processes (first, formation of intrapolymer complexes and then formation of interpolymer complexes), both driven by favorable electrostatic interactions between the negatively charged sulfonic groups (-SO3-) of NaPSS and positively charged patches on the BSA surface. Two such positive patches were identified, each responsible for one of the two binding processes. The presence of salts screened both short-range attractive and long-range repulsive electrostatic interactions between both macromolecules, resulting in a nonmonotonic dependence of the binding affinity on the total ionic strength for both binding processes. In addition, distinct anion-specific effects were observed (NaCl < NaBr < NaI). The effect of sugars was less pronounced: sucrose had no effect on the complexation, but its chlorinated analogue, sucralose, promoted it slightly due to the screening of long-range repulsive electrostatic interactions between BSA and NaPSS. Although short-range non-electrostatic interactions are frequently mentioned in the literature in relation to BSA or NaPSS, we found that the main driving force of complexation on the "wrong side" are electrostatic interactions.

Central European Institute of Technology Masaryk University Kamenice 5 CZ 62500 Brno Czechia

Department of Chemistry Faculty of Science Masaryk University Kamenice 5 CZ 62500 Brno Czechia

Faculty of Chemistry and Chemical Technology University of Ljubljana Večna Pot 113 SI 1000 Ljubljana Slovenia

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Xiao Z.; Liu W.; Zhu G.; Zhou R.; Niu Y. A Review of the Preparation and Application of Flavour and Essential Oils Microcapsules Based on Complex Coacervation Technology. J. Sci. Food Agric. 2014, 94, 1482–1494. 10.1002/jsfa.6491. PubMed DOI

Wagoner T.; Vardhanabhuti B.; Foegeding E. A. Designing Whey Protein–Polysaccharide Particles for Colloidal Stability. Annu. Rev. Food Sci. Technol. 2016, 7, 93–116. 10.1146/annurev-food-041715-033315. PubMed DOI

Zhao L.; Skwarczynski M.; Toth I. Polyelectrolyte-Based Platforms for the Delivery of Peptides and Proteins. ACS Biomater. Sci. Eng. 2019, 5, 4937–4950. 10.1021/acsbiomaterials.9b01135. PubMed DOI

Zheng K.; Chen Y.; Wang X.; Zhao X.; Qian W.; Xu Y. Selective Protein Separation Based on Charge Anisotropy by Spherical Polyelectrolyte Brushes. Langmuir 2020, 36, 10528–10536. 10.1021/acs.langmuir.0c01802. PubMed DOI

Xu Y.; Mazzawi M.; Chen K.; Sun L.; Dubin P. L. Protein Purification by Polyelectrolyte Coacervation: Influence of Protein Charge Anisotropy on Selectivity. Biomacromolecules 2011, 12, 1512–1522. 10.1021/bm101465y. PubMed DOI

Lente J. J.; Lindhoud S. Extraction of Lysozyme from Chicken Albumen Using Polyelectrolyte Complexes. Small 2021, 18, 2105147.10.1002/smll.202105147. PubMed DOI

van Lente J. J.; Claessens M. M. A. E.; Lindhoud S. Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles. Biomacromolecules 2019, 20, 3696–3703. 10.1021/acs.biomac.9b00701. PubMed DOI PMC

Banani S. F.; Lee H. O.; Hyman A. A.; Rosen M. K. Biomolecular Condensates: Organizers of Cellular Biochemistry. Nat. Rev. Mol. Cell Biol. 2017, 18, 285–298. 10.1038/nrm.2017.7. PubMed DOI PMC

Zhou H.; Mangelsdorf M.; Liu J.; Zhu L.; Wu J. Y. RNA-Binding Proteins in Neurological Diseases. Sci. China Life Sci. 2014, 57, 432–444. 10.1007/s11427-014-4647-9. PubMed DOI

Khalil A. M.; Rinn J. L. RNA–Protein Interactions in Human Health and Disease. Semin. Cell Dev. Biol. 2011, 22, 359–365. 10.1016/j.semcdb.2011.02.016. PubMed DOI PMC

Korolev N.; Allahverdi A.; Lyubartsev A. P.; Nordenskiöld L. The Polyelectrolyte Properties of Chromatin. Soft Matter 2012, 8, 9322–9333. 10.1039/c2sm25662b. DOI

Cooper C. L.; Dubin P. L.; Kayitmazer A. B.; Turksen S. Polyelectrolyte–Protein Complexes. Curr. Opin. Colloid Interface Sci. 2005, 10, 52–78. 10.1016/j.cocis.2005.05.007. DOI

Kulkarni A. D.; Vanjari Y. H.; Sancheti K. H.; Patel H. M.; Belgamwar V. S.; Surana S. J.; Pardeshi C. V. Polyelectrolyte Complexes: Mechanisms, Critical Experimental Aspects, and Applications. Artif. Cell Nanomed. Biotechnol. 2016, 44, 1615–1625. 10.3109/21691401.2015.1129624. PubMed DOI

Meka V. S.; Sing M. K. G.; Pichika M. R.; Nali S. R.; Kolapalli V. R. M.; Kesharwani P. A Comprehensive Review on Polyelectrolyte Complexes. Drug Discov. 2017, 22, 1697–1706. 10.1016/j.drudis.2017.06.008. PubMed DOI

Kayitmazer A. B. Thermodynamics of Complex Coacervation. Adv. Colloid Interface Sci. 2017, 239, 169–177. 10.1016/j.cis.2016.07.006. PubMed DOI

Achazi K.; Haag R.; Ballauff M.; Dernedde J.; Kizhakkedathu J. N.; Maysinger D.; Multhaup G. Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems. Angew. Chem., Int. Ed. 2021, 60, 3882–3904. 10.1002/anie.202006457. PubMed DOI PMC

Park J. M.; Muhoberac B. B.; Dubin P. L.; Xia J. Effects of Protein Charge Heterogeneity in Protein-Polyelectrolyte Complexation. Macromolecules 1992, 25, 290–295. 10.1021/ma00027a047. DOI

Mattison K. W.; Dubin P. L.; Brittain I. J. Complex Formation Between Bovine Serum Albumin and Strong Polyelectrolytes: Effect of Polymer Charge Density. J. Phys. Chem. B 1998, 102, 3830–3836. 10.1021/jp980486u. DOI

Yigit C.; Heyda J.; Ballauff M.; Dzubiella J. Like-Charged Protein-Polyelectrolyte Complexation Driven by Charge Patches. J. Chem. Phys. 2015, 143, 064905.10.1063/1.4928078. PubMed DOI

da Silva F. L. B.; Lund M.; Jönsson B.; Åkesson T. On the Complexation of Proteins and Polyelectrolytes. J. Phys. Chem. B 2006, 110, 4459–4464. 10.1021/jp054880l. PubMed DOI

Hattori T.; Hallberg R.; Dubin P. L. Roles of Electrostatic Interaction and Polymer Structure in the Binding of β-Lactoglobulin to Anionic Polyelectrolytes: Measurement of Binding Constants by Frontal Analysis Continuous Capillary Electrophoresis. Langmuir 2000, 16, 9738–9743. 10.1021/la000648p. DOI

Seyrek E.; Dubin P. L.; Tribet C.; Gamble E. A. Ionic Strength Dependence of Protein-Polyelectrolyte Interactions. Biomacromolecules 2003, 4, 273–282. 10.1021/bm025664a. PubMed DOI

de Kruif C. G.; Weinbreck F.; de Vries R. Complex Coacervation of Proteins and Anionic Polysaccharides. Curr. Opin. Colloid Interface Sci. 2004, 9, 340–349. 10.1016/j.cocis.2004.09.006. DOI

Grymonpré K. R.; Staggemeier B. A.; Dubin P. L.; Mattison K. W. Identification by Integrated Computer Modeling and Light Scattering Studies of an Electrostatic Serum Albumin-Hyaluronic Acid Binding Site. Biomacromolecules 2001, 2, 422.10.1021/bm005656z. PubMed DOI

da Silva F. L. B.; Jönsson B. Polyelectrolyte–Protein Complexation Driven by Charge Regulation. Soft Matter 2009, 5, 2862–2868. 10.1039/b902039j. DOI

Lund M.; Jönsson B. Charge Regulation in Biomolecular Solution. Q. Rev. Biophys. 2013, 46, 265–281. 10.1017/s003358351300005x. PubMed DOI

Teramoto A.; Watanabe M.; Iizuka E.; Abe K. Interaction of Polyelectrolytes with Albumin Using Fluorescence Measurement. J. Macromol. Sci. A 1994, 31, 53–64. 10.1080/10601329408545258. DOI

Tribet C.; Porcar I.; Bonnefont P. A.; Audebert R. Association Between Hydrophobically Modified Polyanions and Negatively Charged Bovine Serum Albumin. J. Phys. Chem. B 1998, 102, 1327–1333. 10.1021/jp973022p. DOI

Gao Y.Binding of Proteins to Polyelectrolytes Studied by Capillary Electrophoresis. Ph.D. Thesis, Purdue University, 1998.

Gao J. Y.; Dubin P. L.; Muhoberac B. B. Capillary Electrophoresis and Dynamic Light Scattering Studies of Structure and Binding Characteristics of Protein- Polyelectrolyte Complexes. J. Phys. Chem. B 1998, 102, 5529–5535. 10.1021/jp980507k. DOI

Salis A.; Boström M.; Medda L.; Cugia F.; Barse B.; Parsons D. F.; Ninham B. W.; Monduzzi M. Measurements and Theoretical Interpretation of Points of Zero Charge/Potential of BSA Protein. Langmuir 2011, 27, 11597–11604. 10.1021/la2024605. PubMed DOI

Simončič M.; Lukšič M. Modulating Role of Co-Solutes in Complexation between Bovine Serum Albumin and Sodium Polystyrene Sulfonate. Polymers 2022, 14, 1245.10.3390/polym14061245. PubMed DOI PMC

Bukala J.; Yavvari P.; Walkowiak J. J.; Ballauff M.; Weinhart M. Interaction of Linear Polyelectrolytes with Proteins: Role of Specific Charge–Charge Interaction and Ionic Strength. Biomolecules 2021, 11, 1377.10.3390/biom11091377. PubMed DOI PMC

Yu S.; Xu X.; Yigit C.; van der Giet M.; Zidek W.; Jankowski J.; Dzubiella J.; Ballauff M. Interaction of Human Serum Albumin with Short Polyelectrolytes: A Study by Calorimetry and Computer Simulations. Soft Matter 2015, 11, 4630–4639. 10.1039/c5sm00687b. PubMed DOI

Vinayahan T.; Williams P. A.; Phillips G. O. Electrostatic Interaction and Complex Formation Between Gum Arabic and Bovine Serum Albumin. Biomacromolecules 2010, 11, 3367–3374. 10.1021/bm100486p. PubMed DOI

Walkowiak J. J.; Ballauff M.; Zimmermann R.; Freudenberg U.; Werner C. Thermodynamic Analysis of the Interaction of Heparin With Lysozyme. Biomacromolecules 2020, 21, 4615–4625. 10.1021/acs.biomac.0c00780. PubMed DOI

Aberkane L.; Jasniewski J.; Gaiani C.; Scher J.; Sanchez C. Thermodynamic Characterization of Acacia Gum-β-Lactoglobulin Complex Coacervation. Langmuir 2010, 26, 12523–12533. 10.1021/la100705d. PubMed DOI

Girard M.; Turgeon S. L.; Gauthier S. F. Thermodynamic Parameters of β-Lactoglobulin-Pectin Complexes Assessed by Isothermal Titration Calorimetry. J. Agric. Food Chem. 2003, 51, 4450–4455. 10.1021/jf0259359. PubMed DOI

Wittemann A.; Haupt B.; Ballauff M. Adsorption of Proteins on Spherical Polyelectrolyte Brushes in Aqueous Solution. Phys. Chem. Chem. Phys. 2003, 5, 1671–1677. 10.1039/b300607g. PubMed DOI

Henzler K.; Haupt B.; Lauterbach K.; Wittemann A.; Borisov O.; Ballauff M. Adsorption of β-Lactoglobulin on Spherical Polyelectrolyte Brushes: Direct Proof of Counterion Release by Isothermal Titration Calorimetry. J. Am. Chem. Soc. 2010, 132, 3159–3163. 10.1021/ja909938c. PubMed DOI

Walkowiak J.; Lu Y.; Gradzielski M.; Zauscher S.; Ballauff M. Thermodynamic Analysis of the Uptake of a Protein in a Spherical Polyelectrolyte Brush. Macromol. Rapid Commun. 2020, 41, 1900421.10.1002/marc.201900421. PubMed DOI

Antonov M.; Mazzawi M.; Dubin P. L. Entering and Exiting the Protein-Polyelectrolyte Coacervate Phase via Monmonotonic Salt Dependence of Critical Conditions. Biomacromolecules 2010, 11, 51–59. 10.1021/bm900886k. PubMed DOI

Record M. T.; Anderson C. F.; Lohman T. M. Thermodynamic Analysis of Ion Effects on the Binding and Conformational Equilibria of Proteins and Nucleic Acids: The Roles of Ion Association or Release, Screening, and Ion Effects on Water Activity. Q. Rev. Biophys. 1978, 11, 103–178. 10.1017/s003358350000202x. PubMed DOI

Spruijt E.Strength, Structure and Stability of Polyelectrolyte Complex Coacervates. Ph.D. Thesis, Wageningen University, 2012.

Mimura M.; Tsumura K.; Matsuda A.; Akatsuka N.; Shiraki K. Effect of Additives on Liquid Droplet of Protein–Polyelectrolyte Complex for High-Concentration Formulations. J. Chem. Phys. 2019, 150, 064903.10.1063/1.5063378. PubMed DOI

Wang Y.; Annunziata O. Comparison between Protein-Polyethylene Glycol (PEG) Interactions and the Effect of PEG on Protein-Protein Interactions Using the Liquid-Liquid Phase Transition. J. Phys. Chem. B 2007, 111, 1222–1230. 10.1021/jp065608u. PubMed DOI

Yamanaka J.; Matsuoka H.; Kitano H.; Hasegawa M.; Ise N. Revisit to the Intrinsic Viscosity-Molecular Weight Relationship of Ionic Polymers. 2. Viscosity Behavior of Salt-Free Aqueous Solutions of Sodium Poly(styrenesulfonates). J. Am. Chem. Soc. 1990, 112, 587–592. 10.1021/ja00158a015. DOI

Keller S.; Vargas C.; Zhao H.; Piszczek G.; Brautigam C. A.; Schuck P. High-Precision Isothermal Titration Calorimetry with Automated Peak-Shape Analysis. Anal. Chem. 2012, 84, 5066–5073. 10.1021/ac3007522. PubMed DOI PMC

Bončina M.; Lah J.; Reščič J.; Vlachy V. Thermodynamics of the Lysozyme-Salt Interaction from Calorimetric Titrations. J. Phys. Chem. B 2010, 114, 4313–4319. PubMed

Freire E.; Mayorga O. L.; Straume M. Isothermal Titration Calorimetry. Anal. Chem. 1990, 62, 950A–959A. 10.1021/ac00217a002. DOI

Lakowicz J. R.Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2013.

Anand U.; Mukherjee S. Reversibility in protein folding: effect of β-cyclodextrin on bovine serum albumin unfolded by sodium dodecyl sulphate. Phys. Chem. Chem. Phys. 2013, 15, 9375–9383. 10.1039/c3cp50207d. PubMed DOI

Raut S.; Chib R.; Butler S.; Borejdo J.; Gryczynski Z.; Gryczynski I. Evidence of energy transfer from tryptophan to BSA/HSA protected gold nanoclusters. Methods Appl. Fluoresc. 2014, 2, 035004.10.1088/2050-6120/2/3/035004. PubMed DOI

Micsonai A.; Wien F.; Kernya L.; Lee Y.-H.; Goto Y.; Réfrégiers M.; Kardos J. Accurate Secondary Structure Prediction and Fold Recognition for Circular Dichroism Spectroscopy. Proc. Natl. Acad. Sci. USA 2015, 112, E309510.1073/pnas.1500851112. PubMed DOI PMC

Bujacz A. Structures of Bovine, Equine and Leporine Serum Albumin. Acta Crystallogr. D 2012, 68, 1278–1289. 10.1107/s0907444912027047. PubMed DOI

Dolinsky T. J.; Nielsen J. E.; McCammon J. A.; Baker N. A. PDB2PQR: An Automated Pipeline for the Setup of Poisson–Boltzmann Electrostatics Calculations. Nucleic Acids Res. 2004, 32, W665–W667. 10.1093/nar/gkh381. PubMed DOI PMC

Pahari S.; Sun L.; Basu S.; Alexov E. DelPhiPKa: Including Salt in the Calculations and Enabling Polar Residues to Titrate. Proteins 2018, 86, 1277–1283. 10.1002/prot.25608. PubMed DOI PMC

Pettersen E. F.; Goddard T. D.; Huang C. C.; Couch G. S.; Greenblatt D. M.; Meng E. C.; Ferrin T. E. UCSF Chimera – A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25, 1605–1612. 10.1002/jcc.20084. PubMed DOI

Kyte J.; Doolittle R. F. A Simple Method for Displaying the Hydropathic Character of a Protein. J. Mol. Biol. 1982, 157, 105–132. 10.1016/0022-2836(82)90515-0. PubMed DOI

Tainaka K. Study of Complex Coacervation in Low Concentration by Virial Expansion Method. I. Salt Free Systems. J. Phys. Soc. Jpn. 1979, 46, 1899–1906. 10.1143/jpsj.46.1899. DOI

Chodankar S.; Aswal V. K.; Kohlbrecher J.; Vavrin R.; Wagh A. G. Structural Study of Coacervation in Protein-Polyelectrolyte Complexes. Phys. Rev. E 2008, 78, 031913.10.1103/physreve.78.031913. PubMed DOI

Lunkad R.; Barroso da Silva F. L.; Košovan P. Both Charge-Regulation and Charge-Patch Distribution Can Drive Adsorption on the Wrong Side of the Isoelectric Point. J. Am. Chem. Soc. 2022, 144, 1813–1825. 10.1021/jacs.1c11676. PubMed DOI

Yadav S.; Shire S. J.; Kalonia D. S. Viscosity Analysis of High Concentration Bovine Serum Albumin Aqueous Solutions. Pharm. Res. 2011, 28, 1973–1983. 10.1007/s11095-011-0424-7. PubMed DOI

Takashima S. A. Study of Proton Fluctuation in Protein. Experimental Study of the Kirkwood-Shumaker Theory. J. Phys. Chem. 1965, 69, 2281–2286. 10.1021/j100891a023. DOI

Zaidi N.; Ajmal M. R.; Rabbani G.; Ahmad E.; Khan R. H. A Comprehensive Insight into Binding of Hippuric Acid to Human Serum Albumin: A Study to Uncover Its Impaired Elimination through Hemodialysis. PLoS One 2013, 8, e7142210.1371/journal.pone.0071422. PubMed DOI PMC

Peters T., Jr.All about Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, 1996.

Bowman W. A.; Rubinstein M.; Tan J. S. Polyelectrolyte-Gelatin Complexation: Light-Scattering Study. Macromolecules 1997, 30, 3262–3270. 10.1021/ma961915u. DOI

Hattori T.; Bat-Aldar S.; Kato R.; Bohidar H. B.; Dubin P. L. Characterization of Polyanion–Protein Complexes by Frontal Analysis Continuous Capillary Electrophoresis and Small Angle Neutron Scattering: Effect of Polyanion Flexibility. Anal. Biochem. 2005, 342, 229–236. 10.1016/j.ab.2005.03.043. PubMed DOI

Hallberg R. K.; Dubin P. L. Effect of pH on the Binding of β-Lactoglobulin to Sodium Polystyrenesulfonate. J. Phys. Chem. B 1998, 102, 8629–8633. 10.1021/jp982745l. DOI

Ueberbacher R.; Haimer E.; Hahn R.; Jungbauer A. Hydrophobic Interaction Chromatography of Proteins: V. Quantitative Assessment of Conformational Changes. J. Chromatogr., A 2008, 1198–1199, 154–163. 10.1016/j.chroma.2008.05.062. PubMed DOI

Rodler A.; Beyer B.; Ueberbacher R.; Hahn R.; Jungbauer A. Hydrophobic Interaction Chromatography of Proteins: Studies of Unfolding upon Adsorption by Isothermal Titration Calorimetry. J. Sep. Sci. 2018, 41, 3069–3080. 10.1002/jssc.201800016. PubMed DOI PMC

Tricot M. Comparison of Experimental and Theoretical Persistence Length of some Polyelectrolytes at Various Ionic Strengths. Macromolecules 1984, 17, 1698–1704. 10.1021/ma00139a010. DOI

Spiteri M. N.; Boué F.; Lapp A.; Cotton J. P. Persistence Length for a PSSNa Polyion in Semidilute Solution as a Function of the Ionic Strength. Phys. Rev. Lett. 1996, 77, 5218.10.1103/physrevlett.77.5218. PubMed DOI

Schwierz N.; Horinek D.; Sivan U.; Netz R. R. Reversed Hofmeister Series-The Rule Rather Than the Exception. Curr. Opin. Colloid Interface Sci. 2016, 23, 10–18. 10.1016/j.cocis.2016.04.003. DOI

Janc T.; Vlachy V.; Lukšič M. Calorimetric Studies of Interactions Between Low Molecular Weight Salts and Bovine Serum Albumin in Water at pH Values Below and Above the Isoionic Point. J. Mol. Liq. 2018, 270, 74–80. 10.1016/j.molliq.2017.10.105. PubMed DOI PMC

Simončič M.; Lukšič M. Mechanistic Differences in the Effects of Sucrose and Sucralose on the Phase Stability of Lysozyme Solutions. J. Mol. Liq. 2021, 326, 115245.10.1016/j.molliq.2020.115245. PubMed DOI PMC