title: High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: a single-molecule study.
abstract: The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA. Electronic address: lk12@rice.edu.
authors.forename: Lydia
authors.initials: L
authors.lastname: Kisley
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA. Electronic address: Jixin.chen@rice.edu.
authors.forename: Jixin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA. Electronic address: andrea.p.mansur@rice.edu.
authors.forename: Andrea P
authors.initials: AP
authors.lastname: Mansur
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA. Electronic address: Sergio.Dominguez-Medina@rice.edu.
authors.forename: Sergio
authors.initials: S
authors.lastname: Dominguez-Medina
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA. Electronic address: Eliona.Kulla@rice.edu.
authors.forename: Eliona
authors.initials: E
authors.lastname: Kulla
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA. Electronic address: Marci.Kang@rice.edu.
authors.forename: Marci K
authors.initials: MK
authors.lastname: Kang
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA. Electronic address: bs16@rice.edu.
authors.forename: Bo
authors.initials: B
authors.lastname: Shuang
authors.affiliation: Department of Chemical & Biomolecular Engineering, University of Houston, Houston, TX 77004, USA. Electronic address: edkourentzi@uh.edu.
authors.forename: Katerina
authors.initials: K
authors.lastname: Kourentzi
authors.affiliation: Department of Biology & Biochemistry, University of Houston, Houston, TX 77004, USA. Electronic address: mohan.vivekanandha@gmail.com.
authors.forename: Mohan-Vivekanandan
authors.initials: MV
authors.lastname: Poongavanam
authors.affiliation: Department of Biology & Biochemistry, University of Houston, Houston, TX 77004, USA. Electronic address: dhamane.edu@gmail.com.
authors.forename: Sagar
authors.initials: S
authors.lastname: Dhamane
authors.affiliation: Department of Chemical & Biomolecular Engineering, University of Houston, Houston, TX 77004, USA; Department of Biology & Biochemistry, University of Houston, Houston, TX 77004, USA; Houston Methodist Research Institute, Houston, TX 77030, USA; Centro de Biotecnología FEMSA, Departamento de Biotecnología e Ingeniería de Alimentos, Tecnológico de Monterrey, Monterrey NL 64849, Mexico. Electronic address: willson@uh.edu.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Willson
authors.affiliation: Department of Chemistry, Rice University, Houston, TX 77251, USA; Department of Electrical and Computer Engineering, Rice University, Houston, TX 77251, USA. Electronic address: cflandes@rice.edu.
authors.forename: Christy F
authors.initials: CF
authors.lastname: Landes
mesh_terms: D000327:Adsorption; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002942:Circular Dichroism; D007700:Kinetics; D007768:Lactalbumin; D008024:Ligands; D009994:Osmolar Concentration; D011506:Proteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry
chemical_list: D008024:Ligands; D011506:Proteins; D012685:Sepharose; D007768:Lactalbumin
keywords: Bioseparations; Heterogeneity; Ion-exchange; Optical nanoscopy; mbPAINT
references.citation: J Chromatogr A. 2007 May 25;1150(1-2):259-66
references.pmid: 17054967
references.citation: Langmuir. 2007 Feb 13;23(4):1948-52
references.pmid: 17279679
references.citation: J Chromatogr A. 2007 Aug 10;1160(1-2):206-14
references.pmid: 17560582
references.citation: J Chromatogr A. 1994 Oct 28;684(1):55-63
references.pmid: 7987477
references.citation: Nucleic Acids Res. 2000 Jun 15;28(12):E66
references.pmid: 10871389
references.citation: Biochemistry. 2007 Dec 4;46(48):13864-73
references.pmid: 17985929
references.citation: Analyst. 2012 Jul 7;137(13):2987-96
references.pmid: 22617120
references.citation: Acc Chem Res. 2012 Nov 20;45(11):1927-35
references.pmid: 23013103
references.citation: J Chromatogr A. 2007 May 18;1149(2):312-20
references.pmid: 17439816
references.citation: Anal Chem. 2005 Apr 1;77(7):2157-65
references.pmid: 15801750
references.citation: Biochim Biophys Acta. 1995 Mar 15;1247(2):293-7
references.pmid: 7696322
references.citation: Anal Chem. 2005 Apr 15;77(8):2524-35
references.pmid: 15828789
references.citation: J Chromatogr. 1990 May 4;505(2):329-47
references.pmid: 2355064
references.citation: Anal Chem. 2002 Jan 15;74(2):386-93
references.pmid: 11811413
references.citation: Anal Chem. 2012 Sep 4;84(17):7367-73
references.pmid: 22803776
references.citation: J Chromatogr A. 2006 Sep 8;1126(1-2):107-19
references.pmid: 16844131
references.citation: J Chromatogr A. 2006 Sep 8;1126(1-2):257-67
references.pmid: 16815427
references.citation: J Mol Recognit. 2012 Aug;25(8):435-42
references.pmid: 22811068
references.citation: J Chromatogr A. 2005 Jun 24;1079(1-2):116-26
references.pmid: 16038297
references.citation: J Chromatogr A. 1998 Dec 11;827(2):281-93
references.pmid: 9914656
references.citation: Langmuir. 2006 Dec 19;22(26):11148-57
references.pmid: 17154596
references.citation: Anal Chem. 2010 Jan 1;82(1):189-96
references.pmid: 19957961
references.citation: Anal Biochem. 1983 Aug;133(1):251-9
references.pmid: 6638483
references.citation: Protein Sci. 1999 Mar;8(3):554-61
references.pmid: 10091658
references.citation: J Chromatogr A. 2009 Mar 27;1216(13):2612-5
references.pmid: 19211103
references.citation: J Chromatogr A. 2009 May 15;1216(20):4465-74
references.pmid: 19342054
references.citation: Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):2075-80
references.pmid: 24459184
references.citation: J Chromatogr A. 2011 Jan 14;1218(2):258-62
references.pmid: 21159348
references.citation: Biotechnol Bioeng. 2005 Jul 20;91(2):139-53
references.pmid: 15889407
references.citation: Biophys J. 1998 Apr;74(4):2036-45
references.pmid: 9545062
references.citation: Annu Rev Phys Chem. 2007;58:489-510
references.pmid: 17105411
references.citation: J Phys Chem B. 2009 Nov 5;113(44):14611-8
references.pmid: 19813742
references.citation: Methods Appl Fluoresc. 2013 Sep 1;1(3):037001-37001
references.pmid: 24587894
references.citation: Anal Chem. 2002 Dec 15;74(24):6334-9
references.pmid: 12510756
references.citation: J Chromatogr A. 2009 May 15;1216(20):4372-6
references.pmid: 19168177
references.citation: Anal Chem. 2007 Dec 1;79(23):9060-5
references.pmid: 17973458
references.citation: Anal Chem. 2013 Oct 1;85(19):9363-70
references.pmid: 23998479
references.citation: J Chromatogr A. 2013 Apr 12;1285:78-87
references.pmid: 23489486
references.citation: Anal Chem. 2001 Mar 15;73(6):1091-9
references.pmid: 11305636
references.citation: ACS Appl Mater Interfaces. 2013 Oct 9;5(19):9338-43
references.pmid: 24073628
references.citation: Biotechnol Bioeng. 1995 Dec 5;48(5):437-43
references.pmid: 18623507