Jyotsana Pathak

Interactions in globular proteins with polyampholyte: coacervation route for protein separation

In this work, we report exclusive separation of Bovine Serum Albumin (BSA) from a solution where this protein was present with β-lactoglobulin (β-Lg) in 1 : 0.75 (w/v) ratio at their common isoelectric pH (5 ± 0.02). A polyampholytic polypeptide Gelatin B (GB) also having the same pI was used to extract protein (BSA or β-Lg) molecules selectively from this solution through a process called complex coacervation. In our study, the protein-rich condensate, called coacervate, comprised of GB–BSA complexes while the supernatant mostly contained β-Lg molecules. For the separation of BSA from BSA–GB coacervate, we used ethyl alcohol, which removed the BSA to the supernatant. The differential binding affinity of BSA versus β-Lg to GB chains was established through fluorescence quenching and fluorescence resonance energy transfer (FRET) studies. The BSA–GB binding protocol followed a surface selective patch binding mechanism and these results were obtained from an array of experimental methods such as UV-vis and fluorescence spectroscopy, small angle neutron scattering (SANS), FTIR and circular dichroism spectroscopy. Herein, it is clearly established that selective coacervation at pI can be used as a method for protein separation.

Surface patch binding and mesophase separation in biopolymeric polyelectrolyte-polyampholyte solutions.

Surface patch binding (SPB) induced mesophase separation causing complex coacervation between biopolymers: gelatin A-gelatin B, chitosan-gelatin A, chitosan-gelatin B, and, agar-gelatin B was investigated with and without salt (I=0-0.3 M NaCl). SPB was induced by pH change and three characteristic pHs identified transitions in a turbidity plot: intermolecular interactions ensued at pHc, coacervation transition occurred at pHΦ and phase separation was noticed at pHprep. Associative interactions lead to formation of soluble complexes at pHc exclusively through SPB whereas the coacervation transition was driven by electrostatic binding (EB). Neither pHc nor pHΦ displayed discernible ionic strength (till 50 mM) or temperature dependence, but coacervate yield reduced with increase in ionic strength. Coacervation was completely suppressed beyond 50 mM NaCl. Linear combination of attractive and repulsive parts operating between a polyelectrolyte (charged rod) with a polyampholyte (dipole or point charge) was used to model the interaction potential as function of ionic strength. Relative strength of SPB vis a vis EB was used as SPB index to establish a linear relationship with zeta potential ratio of binding partners. Different phase diagrams could be constructed which clearly identified distinct interaction regimes encountered in solutions undergoing coacervation transition.

Hierarchical Surface Charge Dependent Phase States of Gelatin–Bovine Serum Albumin Dispersions Close to Their Common pI

We report interaction between bovine serum albumin ([BSA] = 1% (w/v)) and gelatin B ([GB] = 0.25-3.5% (w/v)) occurring close to their common isoelectric pH (pI). This interaction generated distinguishable multiple soft matter phases like opaque coacervates (phase I) and transparent gels (phase II), where the former are composed of partially charge neutralized intermolecular complexes (zeta potential, ζ ≤ 0) and the latter of overcharged complexes (ζ ≥ 0) that organized into a network pervading the entire sample volume. These phase states were completely governed by the protein mixing ratio r = [GB]:[BSA]. Coacervates, when heated above 32 °C, produced thermoirreversible turbid gels (phase III), stable in the region 32 ≥ T ≤ 50 °C. When the transparent gels were heated to T ≥ 34 °C, these turned into turbid solutions that did form a turbid fragile gel (phase IV) upon cooling. Mechanical and thermal behaviors of aforesaid coacervates (phase I) and gels (phase II) were examined; coacervates had lower storage modulus and melting temperature compared to gels. Cole-Cole plots attributed considerable heterogeneity to coacervate phase, but gels were relatively homogeneous. Raman spectroscopy data suggested differential microenvironment for these phases. Coacervates were mostly hydrated by partially structured water with degree of hydration dependent on gelatin concentration whereas for gels hydration was invariant of [GB]. Small-angle neutron scattering (SANS) data gave static structure factor profiles, I(q), versus wavevector q, that were remarkably different. For transparent gels, data could be split into two distinct regions: (i) 0.01 < q < 0.1 Å(-1), I(q) = IOZ(0)/(1 + q(2)ζgel(2))(2) (Debye-Bueche function) with ζgel = 9-13 nm, and (ii) 0.1 < q < 0.35 Å(-1), I(q) = IOZ(0)/(1 + q(2)ξgel(2)) (Ornstein-Zernike function) with ξgel = 3.1 ± 0.6 nm. Similarly, for coacervate, the aforesaid two q-regions were described by (i) I(q) = IPL(0)q(-α) with α = 1.7 ± 0.1 and (ii) I(q) = IOZ(0)/(1 + q(2)ξcoac(2)) with ξcoac = 1.6 ± 0.2 nm, a value close to the persistence length of gelatin chain (lp ≈ 2 nm). Phase transition from one equilibrium state to another, i.e., phase I to II, was hierarchical in the charge state of the protein-protein complex. Within the same charge state, transition from phase I to III and from phase II to IV was thermally activated. The aforesaid mechanisms are captured in a unique ζ-T phase diagram.

Charge heterogeneity induced binding and phase stability in β-lacto-globulin–gelatin B gels and coacervates at their common pI

An understanding of the interactions between gelatin B (GB) and β-lacto-globulin (β-Lg) mainly arising from surface selective patch binding occurring at their common pI (≈5.0 ± 0.5) in the absence of added salt. Heterogeneous surface charge distribution on β-Lg facilitated such interaction at different mixing ratio ([GB]: [β-Lg] = r) and the GB–β-Lg complexes carried distinctive surface charge (seen through their zeta potential, ζ). For r 1:1 (overcharged regime, ζ > 0) the dispersion remained transparent and homogeneous which gives no phase separation, but the dispersion formed a gel on waiting. The overcharged gels were homogeneous, more rigid and higher melting temperature in comparison to coacervate. In the coacervate phase, the intensity of the scattered light Is, and its time-correlation function [g2(t) − 1] did not evolve with time. In contrast, the gel phase displayed considerable change with aging time tw. For gels, as tw → ∞ the system moved from an ergodic to non-ergodic state. At tw = 0, the correlation function exhibited one relaxation mode due to the system residing deeply inside the ergodic phase and purely mirroring Brownian dynamics. After a characteristic waiting time, tw an additional mode (slow relaxation) appeared which was attributed to inter-chain interaction induced reorganization of entanglements. This characteristic time was the time required for the system to get dynamically arrested, similar observation was made from rheology measurements too. A comprehensive phase diagram depicting the stability of the dispersion in various charged soft matter states of the complex under various temperature conditions was established.

Is surface patch binding between proteins symmetric about isoelectric pH

Surface selective patch binding (SPB) interaction occurring between two protein molecules, bovine serum albumin (BSA) and gelatin B (GB), both having same isoelectric pH (pI ≈ 5) and identical pH-zeta potential profile, was systematically examined. BSA : GB mixing ratio r was varied in the range 0.16–2.00 and ionic strength was varied in the range 0–10 mM, which yielded optimum binding ratio r = 1. The binding profiles produced asymmetric bell-like curves with clearly identifiable pairs of transition pHs: onset of intermolecular interaction, formation of soluble complexes and coalescence of the soluble complexes occurring at pHc1,2, pHφ1,2 and pHm respectively. Since pHm could be approached from either lower or higher side of pI, these profiles yielded pairs of pHc and pHφ values. In fact, we found (pHc2 − pI) > (pHc1 − pI), which clearly indicated that initiation of intermolecular associative interaction was not symmetric about pI (pI = pHm for r ≤ 1, an observation not reported hitherto. Secondly, (pHφ2 − pI) ≈ (pHφ1 − pI) implied that the pH at which soluble complexes formed (pHφ) was always located symmetrically about pHm, irrespective of the binding ratio. Higher binding affinity determined from higher value of pHc2 was confirmed from size measurement results. The change in the turbidity maximum Δτ could be correlated as Δτ ∼ I1/2 implying electrostatic screening of SPB with increase in ionic strength (I). This interaction was modelled using a linear combination of attractive and repulsive electrostatic forces which revealed considerable screening of the interaction potential U, consistent with aforesaid experimental data; ΔU ∼ I1/2. Further, it is concluded that intermolecular binding in protein–polyampholyte systems is qualitatively different from that in protein–polyelectrolyte class.

Hierarchical Internal Structures in Gelatin–Bovine Serum Albumin/β-Lactoglobulin Gels and Coacervates

Herein, we report the comparative study of gels and complex coacervates of bovine serum albumin (BSA) and beta-lactoglobulin (β-Lg) with gelatin close to their common pI. Surface patch binding produced a range of new soft matter phases (interpolymer complexes) such as opaque coacervates (charge neutralized complexes) and transparent gels (overcharged complexes). We emphasize on the comparative study of the microstructure of coacervates and gels formed at different mixing ratios using small angle scattering (SANS) data. It was found that phase states were entirely defined by the mixing ratio r = [GB]:[β-Lg or BSA]. Thermo-viscoelastic profiles of aforesaid samples revealed a smaller storage modulus and lower melting temperature for coacervates compared to gels. Thermally activated samples generated additional phases that were also probed by SANS and rheology. Thus, it is established that intermolecular association between globular proteins and a random coil polypeptide can generate various soft matter states that may facilitate harvesting of novel biomaterials.

Interaction of Globular Plasma Proteins with Water-Soluble CdSe Quantum Dots

The interactions between water-soluble semiconductor quantum dots [hydrophilic 3-mercaptopropionic acid (MPA)-coated CdSe] and three globular plasma proteins, namely, bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and human serum albumin (HSA), are investigated. Acidic residues of protein molecules form electrostatic interactions with these quantum dots (QDs). To determine the stoichiometry of proteins bound to QDs, we used dynamic light scattering (DLS) and zeta potential techniques. Fluorescence resonance energy transfer (FRET) experiments revealed energy transfer from tryptophan residues in the proteins to the QD particles. Quenching of the intrinsic fluorescence of protein molecules was noticed during this binding process (hierarchy HSA<β-Lg <BSA, lower binding affinity for hydrophobic protein molecules). Upon binding with QD particles, the protein molecules underwent substantial conformational changes at the secondary-structure level (50 % helicity lost), due to loss in hydration.

Thermo-reversibility, ergodicity and surface charge–temperature dependent phase diagram of anionic, cationic and neutral co-gels of gelatin–BSA complexes

We have investigated the gelation behavior of polyampholyte gelatin B (GB) in the presence of colloidal plasma protein bovine serum albumin (BSA) as a function of mixing ratio (r = GB : BSA = 1.5, 2, 3, 4), pH (acidic, basic and neutral), and temperature (20–45 °C). Both the bio-molecules have identical isoelectric pH, and similar zeta potential profiles. Formation of a BSA–GB intermolecular complex arising from hydrogen bonding, and surface selective patch binding between the flexible gelatin chain and colloidal protein BSA, with BSA acting as junction zones was probed. We observed the cationic, neutral and anionic gelation occurring at pHs (3, 5 and 7) corresponding to surface charge of the intermolecular complexes (seen through their zeta potential, ζ). The kinetics and dynamics of gelation were observed from the intensity of scattered light, and time-correlation function which evolved with waiting time (tw ≥ tgel), and indicated rapid gelation for pH 5 (neutral gels) compared to other sols at pH 7 (anionic gel) while at pH 3 (cationic gel) very slow gelation was evident. For all gels with a waiting time of tw → ∞ the gels moved from an ergodic to non-ergodic state. At tw = 0 (nascent gel), the correlation function exhibited a single relaxation mode due to the system residing deeply inside the ergodic phase, and mirroring purely Brownian dynamics. After a characteristic waiting time (ergodicity breaking time, τEB), an additional relaxation (slow mode) appeared which was attributed to inter-chain interactions induced by reorganization of entanglements. The rigidity (low frequency storage modulus, G0), and melting temperature Tm of neutral gels was higher than anionic, but was least for cationic gels. Finally, we capture a unique surface charge versus temperature (ζ vs. T) phase diagram at different pHs, and noticed thermally activated phase transitions which indicated multiple self-assembled states of this pair of bio-molecules leading to evolution of several new soft matter phases, all of which were not thermo-reversible.