Co-reporter:Yaxun Fan, Matthias Kellermeier, Amy Y. Xu, Volodymyr Boyko, Sebastian Mirtschin, and Paul L. Dubin
Macromolecules July 25, 2017 Volume 50(Issue 14) pp:5518-5518
Publication Date(Web):July 6, 2017
DOI:10.1021/acs.macromol.7b00584
The onset of soluble complex formation between polycations and nonionic/anionic mixed micelles was found to occur at well-defined micelle surface charge density, σc, which could be modulated via Y, the mole fraction of anionic surfactant in the mixed micelle. Critical values of Y were detected by precision turbidimetry for two polycations, each combined with any of the four mixed micelles formed from two anionic and two nonionic surfactants. The values of Yc observed for each of the resultant eight ternary polycation/anionic–nonionic combinations were used as surrogates for polycation binding affinity: for a given polycation and a given value of Y, micelles with Yc < Y will bind, while those with Yc > Y will not. The polycation affinity of micelles correlated with their “zeta potentials” (ζ), measured by electrophoretic light scattering, and their average surface potentials (ψ0), measured by potentiometric titration of a comicellized probe. For a given polycation at a fixed ionic strength, we found that the critical zeta potential (ζc) measured at Yc was independent of the surfactant pair chosen. This potential at the micelle “shear plane” is thus interpreted as the potential experienced by a bound polycation. The binding affinity was furthermore found to be stronger for polycations with higher linear charge density as well as for micelles with higher axial ratio, attributed respectively to an increase in the number of micelle-bound charged polycation repeat units and to the higher surface potential for micelles with lower surface curvature.
Co-reporter:Burcu Baykal Minsky, Bingqian Zheng, and Paul L. Dubin
Langmuir January 14, 2014 Volume 30(Issue 1) pp:278-287
Publication Date(Web):January 14, 2014
DOI:10.1021/la4039232
Protein native state aggregation, a major problem in pharmaceutical and biological processes, has been addressed pharmacologically by the addition of protein-binding excipients. Heparin (Hp), a highly sulfated polysaccharide, interacts with numerous proteins with moderate to high affinity, but reports about its effect on protein aggregation are contradictory. We studied the pH dependence of the aggregation of antithrombin (AT) and bovine serum albumin (BSA) in the presence and absence of heparin. High-precision turbidimetry showed strong aggregation for both AT and BSA in I = 10 mM NaCl, conditions at which electrostatically driven Hp binding and aggregation both occur, with more obvious aggregation of heparin-free AT appearing as larger aggregate size. Aggregation of AT was dramatically inhibited at Hp: protein 6:1 (mole ratio); however, the effect at 0.5:1 Hp:protein was greater for BSA. Frontal analysis capillary electrophoresis showed a much larger equilibrium association constant Kobs between Hp and AT, in accord with the onset of Hp binding at a higher pH; both effects are explained by the higher charge density of the positive domain for AT as revealed by modeling with DelPhi. The corresponding modeling images showed that these domains persist at high salt only for AT, consistent with the 160-fold drop in Kobs at 100 mM salt for BSA–Hp binding. The smaller inhibition effect for AT arises from the tendency of its uncomplexed monomer to form larger aggregates more rapidly, but the stronger binding of Hp to AT does not facilitate Hp-induced aggregate dissolution which occurs more readily for BSA. This can be attributed to the higher density of AT aggregates evidenced by higher fractal dimensions. Differences between inhibition and reversal by Hp arise because the former may depend on the stage at which Hp enters the aggregation process and the latter on aggregate size and morphology.
Co-reporter:Fatih Comert;Fatemeh Azarikia
Physical Chemistry Chemical Physics 2017 vol. 19(Issue 31) pp:21090-21094
Publication Date(Web):2017/08/09
DOI:10.1039/C7CP02641B
The ζ-potential, a parameter typically obtained by model-dependent transformation of the measured electrophoretic mobility, is frequently used to understand polysaccharide–protein complexation. We tested the hypothesis that two anionic polysaccharides with identical ζ-potentials would show equal binding affinity to the protein β-lactoglobulin (BLG). We selected two polysaccharide polyelectrolytes (PE) with very different structures: hyaluronic acid (HA) and tragacanthin (TG). Highly precise (±0.1%) turbidimetric titrations were performed to determine critical pH values of complex formation; and PE ζ-potentials were measured for different ionic strengths I at those critical pH values. While phase boundaries (pHcvs. I) showed that HA binds to BLG more strongly (e.g. at a lower pH, for fixed I), comparisons made at fixed ζ-potential indicated that TG binds more strongly. The source of this contradiction is the effect of the bulky side chains of TG on its friction coefficient which diminishes its mobility and hence the resultant ζ-potential; while having a distinctly separate effect on the interaction between BLG and the carboxylated backbone of TG. Thus, unless the locus of the bound protein coincides with the shear plane, the ζ-potential does not directly contribute to the electrostatic PE–protein interaction.
Co-reporter:Fatih Comert, Alexander J. Malanowski, Fatemeh Azarikia and Paul L. Dubin
Soft Matter 2016 vol. 12(Issue 18) pp:4154-4161
Publication Date(Web):31 Mar 2016
DOI:10.1039/C6SM00044D
Precipitation poses a consistent problem for the growing applications of biopolymer coacervation, but the relationship between the two types of phase separation is not well understood. To clarify this relationship, we studied phase separation as a function of pH and ionic strength, in three systems of proteins with anionic polysaccharides: β-lactoglobulin (BLG)/hyaluronic acid (HA); BLG/tragacanthin (TG); and monoclonal antibody (mAb)/HA. We found that coacervation and precipitation are intrinsically different phenomena, responsive to different factors, but their simultaneity (for example with changing pH) may be confused with transitions from one state to another. We propose that coacervate does not literally turn into precipitate, but rather that both coacervate and precipitate are in equilibrium with free protein and polyanion, so that dissolution of one and formation of the other can overlap in time. While protein–polyanion complexes must achieve neutrality for coacervation, precipitation only requires tight binding which leads to the expulsion of counterions and water molecules. The pH-dependence of phase separation, considered in terms of protein and polyion charge, revealed that the electrostatic magnitude of the protein's polymer-binding site (“charge patch”) plays a key role in the strength of interaction. These findings were supported by the inhibition of precipitation, seen when the bulky side chains of TG impede close protein–polymer interactions.
Co-reporter:Burcu Baykal Minsky, Bingqian Zheng, and Paul L. Dubin
Langmuir 2014 Volume 30(Issue 1) pp:278-287
Publication Date(Web):2017-2-22
DOI:10.1021/la4039232
Protein native state aggregation, a major problem in pharmaceutical and biological processes, has been addressed pharmacologically by the addition of protein-binding excipients. Heparin (Hp), a highly sulfated polysaccharide, interacts with numerous proteins with moderate to high affinity, but reports about its effect on protein aggregation are contradictory. We studied the pH dependence of the aggregation of antithrombin (AT) and bovine serum albumin (BSA) in the presence and absence of heparin. High-precision turbidimetry showed strong aggregation for both AT and BSA in I = 10 mM NaCl, conditions at which electrostatically driven Hp binding and aggregation both occur, with more obvious aggregation of heparin-free AT appearing as larger aggregate size. Aggregation of AT was dramatically inhibited at Hp: protein 6:1 (mole ratio); however, the effect at 0.5:1 Hp:protein was greater for BSA. Frontal analysis capillary electrophoresis showed a much larger equilibrium association constant Kobs between Hp and AT, in accord with the onset of Hp binding at a higher pH; both effects are explained by the higher charge density of the positive domain for AT as revealed by modeling with DelPhi. The corresponding modeling images showed that these domains persist at high salt only for AT, consistent with the 160-fold drop in Kobs at 100 mM salt for BSA–Hp binding. The smaller inhibition effect for AT arises from the tendency of its uncomplexed monomer to form larger aggregates more rapidly, but the stronger binding of Hp to AT does not facilitate Hp-induced aggregate dissolution which occurs more readily for BSA. This can be attributed to the higher density of AT aggregates evidenced by higher fractal dimensions. Differences between inhibition and reversal by Hp arise because the former may depend on the stage at which Hp enters the aggregation process and the latter on aggregate size and morphology.
Co-reporter:Yisheng Xu, Yoni Engel, Yunfeng Yan, Kaimin Chen, Daniel F. Moyano, Paul L. Dubin and Vincent M. Rotello
Journal of Materials Chemistry A 2013 vol. 1(Issue 39) pp:5230-5234
Publication Date(Web):30 Apr 2013
DOI:10.1039/C3TB20377H
Two β-lactoglobulin (BLG) isoforms, BLGA and BLGB, were used as a test bed for the differentiation of proteins using electrostatics. In these studies, the BLGA and BLGB binding to a highly charged, cationic gold nanoparticle (GNP) modified surface was investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR) spectroscopy. The binding affinity, and more importantly, the selectivity of this surface towards these two almost identical protein isoforms were both significantly increased on the cationic GNP surface array relative to the values measured with the same free cationic GNP in solution. While protein recognition is traditionally achieved almost exclusively via orientation dependent short-range interactions such as hydrogen bonds and hydrophobic interactions, our results show the potential of protein recognition platforms based on enhanced electrostatic interactions.
Co-reporter:Ebru Kizilay, Anthony D. Dinsmore, David A. Hoagland, Lianhong Sun and Paul L. Dubin
Soft Matter 2013 vol. 9(Issue 30) pp:7320-7332
Publication Date(Web):31 May 2013
DOI:10.1039/C3SM50591J
We investigated the temperature-induced liquid–liquid phase separation (coacervation) of polyelectrolyte (PE)–micelle systems and the structure of the resultant coacervates. Dynamic light scattering, small angle neutron scattering and cryo-transmission electron microscopy (DLS, SANS, Cryo-TEM) were used to examine the evolution of complex structure up to the point of temperature-induced coacervation and beyond. Three diffusional modes, seen in the single phase samples and in the coexisting coacervated supernatant phases were attributed respectively to free micelles, PE–micelle complexes, and aggregates thereof. They corresponded to SANS Guinier region slopes yielding Rg ∼ 4 nm (micelles) and Rg ∼ 50 nm (unresolved complexes and aggregates). Cryo-TEM images of coacervates indicated how these subunits are organized within dense and dilute coacervate domains at larger length scales. Taken together, these results are understood to arise from the requirements of overall charge neutralization, and ion-pairing and counterion release during coacervation. We conclude that a polyelectrolyte:micelle system at incipient coacervation with charge stoichiometry ([+]/[−] > 1) donates excess polycations to other complexes in solution. In the coacervate, a similar disproportionation but at different length scales ejects excess polycations and their counterions into dilute domains. In both phases, association and desolvation are driven by counterion release, enhanced chain configurational entropy, and ion-pairing. These enthalpic and entropic forces operating in both phases could explain the structural similarities between soluble aggregates and coacervate dense domains.
Co-reporter:Burcu Baykal Minsky, Thuy V. Nguyen, Shelly R. Peyton, Igor A. Kaltashov, and Paul L. Dubin
Biomacromolecules 2013 Volume 14(Issue 11) pp:
Publication Date(Web):October 9, 2013
DOI:10.1021/bm401227p
Full-length heparin is widely used in tissue engineering applications due its multiple protein-binding sites that allow it to retain growth factor affinity while associating with oligopeptide components of the tissue scaffold. However, the extent to which oligopeptide coupling interferes with cognate protein binding is difficult to predict. To investigate such simultaneous interactions, we examined a well-defined ternary system comprised of acidic fibroblast growth factor (FGF), tetralysine (K4), with a heparin decamer (dp10) acting as a noncovalent coupler. Electrospray ionization mass spectrometry was used to assess binding affinities and complex stoichiometries as a function of ionic strength for dp10·K4 and FGF·dp10. The ionic strength dependence of K4·dp10 formation is qualitatively consistent with binding driven by the release of condensed counterions previously suggested for native heparin with divalent oligopeptides (Mascotti, D. P.; Lohman, T. M. Biochemistry 1995, 34, 2908–2915). On the other hand, FGF binding displays more complex ionic strength dependence, with higher salt resistance. Remarkably, dp10 that can bind two FGF molecules can only bind one tetralysine. The limited binding of K4 to dp10 suggests that the tetralysine might not block growth factor binding, and the 1:1:1 ternary complex is indeed observed. The analysis of mass distribution of the bound dp10 chains in FGF·dp10, FGF2·dp10, and FGF·dp10·K4 complexes indicated that higher degrees of dp10 sulfation promote the formation of FGF2·dp10 and FGF·dp10·K4. Thus, the selectivity of appropriately chosen short heparin chains could be used to modulate growth factor sequestration and release in a way not feasible with heterogeneous native heparin. In support of this, human hepatocellular carcinoma cells (HEP3Bs) treated with FGF·dp10·K4 were found to exhibit biological activity similar to cells treated with FGF.
Co-reporter:Burcu Baykal Minsky, Anand Atmuri, Igor A. Kaltashov, and Paul L. Dubin
Biomacromolecules 2013 Volume 14(Issue 4) pp:
Publication Date(Web):March 4, 2013
DOI:10.1021/bm400006g
The electropherogram of native heparin shows a broad distribution of mobilities μ, which truncates abruptly at a notably high μ = 4.7 × 10–4 cm2 V–1 s–1. This highly skewed mobility distribution is also found for the 20-saccharide chain, which shows from mass spectrometry a more uniform (symmetrical) with respect to sulfation level. Since a partially degraded heparin exhibits oligomer peaks with μ> 5 × 10–4 cm2 V–1 s–1 (appearing to escape the limitation of the mobility value for native heparin), we examined the electrophoretic behavior of chain-length monodisperse heparin oligomers. Their mobilities varied inversely with the logarithm of the contour length, L, for L from 3 to 10 nm and reached an asymptotic limit for L > 20 nm. The generality of this effect was indicated by similar behavior for oligomers of poly(styrene sulfonate). A recent theory of polyelectrolyte end effects (Manning, G. S. Macromolecules2008, 41, 6217–6227), in which chain termini exhibit reduced counterion condensation was found to quantitatively account for these results. A qualitative explanation for the anomalously high value of μ of native heparin, 10–20% higher than those seen for synthetic polyelectrolytes of higher linear charge density, is suggested on the basis of similar junction effects (Manning, G. S. Macromolecules2008, 41, 6217–6227), which reduce counterion condensation at the interfaces of regions of high and low sulfation. We suggest that these effects should be considered in models for the biofunctionality of the regulated high and low sulfation (NS/NA) domains of heparan sulfate.
Co-reporter:Yunfeng Yan, Daniel Seeman, Bingqian Zheng, Ebru Kizilay, Yisheng Xu, and Paul L. Dubin
Langmuir 2013 Volume 29(Issue 14) pp:4584-4593
Publication Date(Web):March 5, 2013
DOI:10.1021/la400258r
The aggregation of β-lactoglobulin (BLG) near its isoelectric point was studied as a function of ionic strength and pH. We compared the behavior of native BLG with those of its two isoforms, BLG-A and BLG-B, and with that of a protein with a very similar pI, bovine serum albumin (BSA). Rates of aggregation were obtained through a highly precise and convenient pH/turbidimetric titration that measures transmittance to ±0.05 %T. A comparison of BLG and BSA suggests that the difference between pHmax (the pH of the maximum aggregation rate) and pI is systematically related to the nature of protein charge asymmetry, as further supported by the effect of localized charge density on the dramatically different aggregation rates of the two BLG isoforms. Kinetic measurements including very short time periods show well-differentiated first and second steps. BLG was analyzed by light scattering under conditions corresponding to maxima in the first and second steps. Dynamic light scattering (DLS) was used to monitor the kinetics, and static light scattering (SLS) was used to evaluate the aggregate structure fractal dimensions at different quench points. The rate of the first step is relatively symmetrical around pHmax and is attributed to the local charges within the negative domain of the free protein. In contrast, the remarkably linear pH dependence of the second step is related to the uniform reduction in global protein charge with increasing pH below pI, accompanied by an attractive force due to surface charge fluctuations.
Co-reporter:Yunfeng Yan, Ebru Kizilay, Daniel Seeman, Sean Flanagan, Paul L. Dubin, Lionel Bovetto, Laurence Donato, and Christophe Schmitt
Langmuir 2013 Volume 29(Issue 50) pp:15614-15623
Publication Date(Web):2017-2-22
DOI:10.1021/la4027464
Lactoferrin (LF) and β-lactoglobulin (BLG), strongly basic and weakly acidic bovine milk proteins, form optically clear coacervates under highly limited conditions of pH, ionic strength I, total protein concentration CP, and BLG:LF stoichiometry. At 1:1 weight ratio, the coacervate composition has the same stoichiometry as its supernatant, which along with DLS measurements is consistent with an average structure LF(BLG2)2. In contrast to coacervation involving polyelectrolytes here, coacervates only form at I < 20 mM. The range of pH at which coacervation occurs is similarly narrow, ca. 5.7–6.2. On the other hand, suppression of coacervation is observed at high CP, similar to the behavior of some polyelectrolyte–colloid systems. It is proposed that the structural homogeneity of complexes versus coacervates with polyelectrolytes greatly reduces the entropy of coacervation (both chain configuration and counterion loss) so that a very precise balance of repulsive and attractive forces is required for phase separation of the coacervate equilibrium state. The liquid–liquid phase transition can however be obscured by the kinetics of BLG aggregation which can compete with coacervation by depletion of BLG.
Co-reporter:Yisheng Xu, Yunfeng Yan, Daniel Seeman, Lianhong Sun, and Paul L. Dubin
Langmuir 2012 Volume 28(Issue 1) pp:579-586
Publication Date(Web):November 7, 2011
DOI:10.1021/la202902a
The aggregation of insulin is complicated by the coexistence of various multimers, especially in the presence of Zn2+. Most investigations of insulin multimerization tend to overlook aggregation kinetics, while studies of insulin aggregation generally pay little attention to multimerization. A clear understanding of the starting multimer state of insulin is necessary for the elucidation of its aggregation mechanism. In this work, the native-state aggregation of insulin as either the Zn–insulin hexamer or the Zn-free dimer was studied by turbidimetry and dynamic light scattering, at low ionic strength and pH near pI. The two states were achieved by varying the Zn2+ content of insulin at low concentrations, in accordance with size-exclusion chromatography results and literature findings (Tantipolphan, R.; Romeijn, S.; Engelsman, J. d.; Torosantucci, R.; Rasmussen, T.; Jiskoot, W. J. Pharm. Biomed. 2010, 52, 195). The much greater aggregation rate and limiting turbidity (τ∞) for the Zn–insulin hexamer relative to the Zn-free dimer was explained by their different aggregation mechanisms. Sequential first-order kinetic regimes and the concentration dependence of τ∞ for the Zn–insulin hexamer indicate a nucleation and growth mechanism, as proposed by Wang and Kurganov (Wang, K.; Kurganov, B. I. Biophys. Chem. 2003, 106, 97). The pure second-order process for the Zn-free dimer suggests isodesmic aggregation, consistent with the literature. The aggregation behavior at an intermediate Zn2+ concentration appears to be the sum of the two processes.
Co-reporter:Yisheng Xu, Daniel Seeman, Yunfeng Yan, Lianhong Sun, Jared Post, and Paul L. Dubin
Biomacromolecules 2012 Volume 13(Issue 5) pp:
Publication Date(Web):April 12, 2012
DOI:10.1021/bm3003539
The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), β-lactoglobulin (BLG), and Zn–insulin at a pH near pI and at low ionic strength I; however, the results vary with regard to the range of pH, I, and protein–heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin.
Co-reporter:Ebru Kizilay, Simona Maccarrone, Elaine Foun, Anthony D. Dinsmore, and Paul L. Dubin
The Journal of Physical Chemistry B 2011 Volume 115(Issue 22) pp:7256-7263
Publication Date(Web):March 28, 2011
DOI:10.1021/jp109788r
The temperature-induced liquid−liquid phase transition (complex coacervation) of a polycation−anionic/nonionic mixed micelle system was examined over a range of macroion concentrations and polycation molecular weights (MW) using turbidimetry and dynamic light scattering (DLS). DLS revealed a progressive increase in complex/aggregate size with temperature up to the phase transition at Tφ, followed by splitting of these clusters into respectively smaller and larger particles. We present two explanations: (1) large (200−400 nm) clusters (soluble aggregates) are necessary and sufficient coacervation precursors, and (2) the process of coacervation itself is accompanied by the expulsion of smaller aggregates to form submicrometer droplets. Although a reduction in Tφ for higher MW appears to be correlated with larger clusters, in support of model 1, the opposite correlation between cluster size and Tφ is seen upon isoionic dilution. We conclude that enhanced coacervation and increased cluster size at high polymer MW arise independently from increased intercomplex attractive forces. Dilution, on the other hand, leads to diminished cluster size, whereas the decrease in Tφ on dilution is a reflection of coacervate self-suppression, previously observed for this system. The splitting of clusters into large and small species near Tφ is explained by macroion disproportionation, as proposed by Shkolvskii et al for DNA condensation. We demonstrate and explain a similar phenomenon: broadening of the phase transition by an increase in cluster polydispersity, resulting from an increase in surfactant polydispersity.
Co-reporter:Yisheng Xu, Malek Mazzawi, Kaimin Chen, Lianhong Sun, and Paul L. Dubin
Biomacromolecules 2011 Volume 12(Issue 5) pp:
Publication Date(Web):March 17, 2011
DOI:10.1021/bm101465y
The effect of polyelectrolyte binding affinity on selective coacervation of proteins with the cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDADMAC), was investigated for bovine serum albumin/β-lactoglobulin (BSA/BLG) and for the isoforms BLG-A/BLG-B. High-sensitivity turbidimetric titrations were used to define conditions of complex formation and coacervation (pHc and pHϕ, respectively) as a function of ionic strength. The resultant phase boundaries, essential for the choice of conditions for selective coacervation for the chosen protein pairs, are nonmonotonic with respect to ionic strength, for both pHc and pHϕ. These results are explained in the context of short-range attraction/long-range repulsion governing initial protein binding “on the wrong side of pI” and also subsequent phase separation due to charge neutralization. The stronger binding of BLG despite its higher isoelectric point, inferred from lower pHc, is shown to result from the negative “charge patch” on BLG, absent for BSA, as visualized via computer modeling (DelPhi). The higher affinity of BLG versus BSA was also confirmed by isothermal titration calorimetry (ITC). The relative values of pHϕ for the two proteins show complex salt dependence so that the choice of ionic strength determines the order of coacervation, whereas the choice of pH controls the yield of the target protein. Coacervation at I = 100 mM, pH 7, of BLG from a 1:1 (w/w) mixture with BSA was shown by SEC to provide 90% purity of BLG with a 20-fold increase in concentration. Ultrafiltration was shown to remove effectively the polymer from the target protein. The relationship between protein charge anisotropy and binding affinity and between binding affinity and selective coacervation, inferred from the results for BLG/BSA, was tested using the isoforms of BLG. Substitution of glycine in BLG-B by aspartate in BLG-A lowers pHc by 0.2, as anticipated on the basis of DelPhi modeling. The stronger binding of BLG-A, confirmed by ITC, led to a difference in pHϕ that was sufficient to provide enrichment by a factor of 2 for BLG-A in the coacervate formed from “native BLG”.
Co-reporter:Kaimin Chen, Yisheng Xu, Subinoy Rana, Oscar R. Miranda, Paul L. Dubin, Vincent M. Rotello, Lianhong Sun, and Xuhong Guo
Biomacromolecules 2011 Volume 12(Issue 7) pp:
Publication Date(Web):May 17, 2011
DOI:10.1021/bm200374e
The binding of bovine serum albumin (BSA) and β-lactoglobulin (BLG) to TTMA (a cationic gold nanoparticle coupled to 3,6,9,12-tetraoxatricosan-1-aminium, 23-mercapto-N,N,N-trimethyl) was studied by high-resolution turbidimetry (to observe a critical pH for binding), dynamic light scattering (to monitor particle growth), and isothermal titration calorimetry (to measure binding energetics), all as a function of pH and ionic strength. Distinctively higher affinities observed for BLG versus BSA, despite the lower pI of the latter, were explained in terms of their different charge anisotropies, namely, the negative charge patch of BLG. To confirm this effect, we studied two isoforms of BLG that differ in only two amino acids. Significantly stronger binding to BLGA could be attributed to the presence of the additional aspartates in the negative charge domain for the BLG dimer, best portrayed in DelPhi. This selectivity decreases at low ionic strength, at which both isoforms bind well below pI. Selectivity increases with ionic strength for BLG versus BSA, which binds above pI. This result points to the diminished role of long-range repulsions for binding above pI. Dynamic light scattering reveals a tendency for higher-order aggregation for TTMA–BSA at pH above the pI of BSA, due to its ability to bridge nanoparticles. In contrast, soluble BLG–TTMA complexes were stable over a range of pH because the charge anisotropy of this protein at makes it unable to bridge nanoparticles. Finally, isothermal titration calorimetry shows endoenthalpic binding for all proteins: the higher affinity of TTMA for BLGA versus BLGB comes from a difference in the dominant entropy term.
Co-reporter:Emek Seyrek, Paul Dubin
Advances in Colloid and Interface Science 2010 Volume 158(1–2) pp:119-129
Publication Date(Web):12 July 2010
DOI:10.1016/j.cis.2010.03.001
Abstract
One of the barriers to understanding structure–property relations for glycosaminoglycans has been the lack of constructive interplay between the principles and methodologies of the life sciences (molecular biology, biochemistry and cell biology) and the physical sciences, particularly in the field of polyelectrolytes. To address this, we first review the similarities and differences between the physicochemical properties of GAGs and other statistical chain polyelectrolytes of both natural and abioitic origin. Since the biofunctionality and regulation of the structures of GAGs is intimately connected with interactions with their cognate proteins, we particularly compare and contrast aspects of protein binding, i.e. effects of both GAGs and other polyelectrolytes on protein stability, protein aggregation and phase behavior. The protein binding affinities and their dependences on pH and ionic strength for the two groups are discussed not only in terms of observable differences, but also with regard to contrasting descriptions of the bound state and the role of electrostatics. We conclude that early studies of the heparin–Antithromin system, proceeding to a large extent through the methods and models of protein chemistry and drug discovery, established not only many enabling precedents but also constraining paradigms. Current studies on heparan sulfate and chondroitin sulfate seem to reflect a more ecumenical view likely to be more compatible with concepts from physical and polymer chemistry.
Co-reporter:Margarita Antonov, Malek Mazzawi and Paul L. Dubin
Biomacromolecules 2010 Volume 11(Issue 1) pp:
Publication Date(Web):November 30, 2009
DOI:10.1021/bm900886k
Critical conditions for coacervation of poly(dimethyldiallylammonium chloride) (PDADMAC) with bovine serum albumin were determined as a function of ionic strength, pH, and protein/polyelectrolyte stoichiometry. The resultant phase boundaries, clearly defined with this narrow molecular weight distribution PDADMAC sample, showed nonmonotonic ionic strength dependence, with the pH-induced onset of coacervation (at pHϕ) occurring most readily at 20 mM NaCl. The corresponding onset of soluble complex formation, pHc, determined using high-precision turbidimetry sensitive to changes of less than 0.1% transmittance units, mirrored the ionic strength dependence of pHϕ. This nonmonotonic binding behavior is attributable to simultaneous screening of short-range attraction and long-range repulsion. The similarity of pHc and pHϕ was explained by the effect of salt on protein binding, and consequently on the number of bound proteins relative to that required for charge neutralization of the complex, a requirement for phase separation. Expansion of the coacervation regime with chitosan, a polycation with charge spacing similar to that of PDADMAC, could be due to either the charge mobility or chain stiffness of the former. The pHϕ versus I phase boundary for PDADMAC correctly predicted entrance into and egress from the coacervation region by addition of either salt or water. The ability to induce or suppress coacervation via protein/polyelectrolyte stoichiometry r was found to be consistent with the proposed model. The results indicate that the conjoint effects of I, r, and pH on coacervation could be represented by a three-dimensional phase boundary.
Co-reporter:Katie Giger, Ram P. Vanam, Emek Seyrek and Paul L. Dubin
Biomacromolecules 2008 Volume 9(Issue 9) pp:
Publication Date(Web):August 13, 2008
DOI:10.1021/bm8002557
The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH- and ionic strength (I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH = pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I = 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.
Co-reporter:Emek Seyrek;Jens Henriksen
Biopolymers 2007 Volume 86(Issue 3) pp:
Publication Date(Web):21 MAR 2007
DOI:10.1002/bip.20731
We evaluated the role of nonspecific electrostatic binding in the interaction of antithrombin (AT) with heparin (Hp), a paradigmatic protein-glycosaminoglycan (GAG) system. To do so, we obtained the ionic-strength dependence of the binding constant, since a common feature in protein-polyelectrolyte systems is a maximum in affinity in the ionic strength range 10 mM <I<30 mM (Seyrek et al, Biomacromolecules 2003, 4, 273-282). Because this feature is seen for both synthetic and biological polyelectrolytes, and because the value of Imax correlates with protein size and charge asymmetry through the Debye length (Seyrek et al, Biomacromolecules 2003, 4, 273-282), this behavior appears to be a signature of non-specific electrostatic protein-polyelectrolyte binding. Binding of AT to both standard (14 kDa) Hp and partially degraded (5 kDa) low molecular weight heparin (LMWH) exhibited this same behavior. Capillary electrophoresis (CZE) of Hp and LMWH yielded electropherograms whose remarkable breadth revealed the enormous heterogeneity of average charge density among the innumerable molecular species of Hp and LMWH. These distributions were somewhat reduced after affinity chromatography (AC) fractionation, indicating that the high-affinity fraction was generally depleted of the lower-charge species. Size-exclusion chromatography coupled with Electrospray Mass Spectrometry confirmed lower levels of sulfation for the lower affinity fractions. Comparisons of LMWH with Dermatan sulfate (DS) by CZE and AC suggested a correlation between the relative absence of very highly charged components in DS and its weaker binding to AT. These findings point to a significant role of the charge density of GAG chains in their affinity for AT. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 249–259, 2007.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Co-reporter:Yisheng Xu, Yoni Engel, Yunfeng Yan, Kaimin Chen, Daniel F. Moyano, Paul L. Dubin and Vincent M. Rotello
Journal of Materials Chemistry A 2013 - vol. 1(Issue 39) pp:NaN5234-5234
Publication Date(Web):2013/04/30
DOI:10.1039/C3TB20377H
Two β-lactoglobulin (BLG) isoforms, BLGA and BLGB, were used as a test bed for the differentiation of proteins using electrostatics. In these studies, the BLGA and BLGB binding to a highly charged, cationic gold nanoparticle (GNP) modified surface was investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR) spectroscopy. The binding affinity, and more importantly, the selectivity of this surface towards these two almost identical protein isoforms were both significantly increased on the cationic GNP surface array relative to the values measured with the same free cationic GNP in solution. While protein recognition is traditionally achieved almost exclusively via orientation dependent short-range interactions such as hydrogen bonds and hydrophobic interactions, our results show the potential of protein recognition platforms based on enhanced electrostatic interactions.
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