Effects of Surface Charge Distribution of Proteins in Their Complexation with Polyelectrolytes in an Aqueous Salt-Free System

Abstract

Complexation between protein and polyelectrolyte was studied by a combination of three main experimental techniques: turbidimetric titration, quasi-elastic light scattering (QELS), and static light scattering (SLS). Proteins of two sorts, lysozyme (Lyz) and ribonuclease (RNase), were chosen by considering the following characteristics: (i) Both proteins have the same number (19) of basic groups; (ii) their distribution is almost homogeneous on Lyz but not on RNase; (iii) there is little difference in the molar mass between both proteins. Potassium poly(vinyl alcohol) sulfate (KPVS) with different molecular weights (M h PE) and various degrees of esterification (De) was used as the polyelectrolyte. We employed a salt-free aqueous medium and adjusted it to pH 2, the level of which forces to completion the protonation of all of the basic groups. As the titration of proteins with KPVS proceeded, the absorbance (A) as an indication of turbidity increased linearly and then rapidly at a certain titrant volume, referring to the end point of titration. The slope of the linear plots of A vs titrant volume for Lyz was little dependent on M h PE and De. In the case of RNase, however, the slope increased with decreasing De and with increasing M h PE. From the studies of the hydrodynamic radius by QELS as well as the molecular weight and radius gyration by SLS, it was found that the M h PE and De effects observed in the titration curve correspond to the changes of both size and mass of aggregated "intrapolymer" complexes formed during titration. Thus, we estimated the degree of aggregation (R) through dividing the mass from SLS by the calculated mass of an intrapolymer complex. This showed that R decreases with increasing De in both protein systems, while increasing M h PE decreases R in the Lyz system but does not change it in the RNase system. These results were discussed in detail by considering the polarizability of the intrapolymer complex, the level of which is affected by the complementarity of the spacing between charges on the protein to the uniform spacing between polymer charges.

Immobilization of lysozyme on poly(N-isopropyl acrylamide)/2-hydroxyethyl methacrylate copolymer core–shell gel beads

“…In previous studies, we examined the complexation of various proteins with water-soluble polymers and the enzymatic activities of the resulting PPCs [1][2][3]. The formed PPCs showed 20-50% of the activity of the native enzymes as has been reported by many researchers.…”

“…Similarly, we reported that the formation mechanism and activity of Lyz complexed with polyelectrolytes were influenced by the molecular size of the substrates (such as M. lysodeikticus and (GluNAc) 6 ), the surface charge density of the Lyz and polyelectrolyte, and the molecular weight of the polyelectrolyte [1][2][3]6]. We therefore next evaluated the relative activity of PGBH-immobilized Lyz with varying HEMA content toward substrates of different molecular sizes for the purpose of determining the efficiency of the PGBH particles as enzyme immobilization carriers.…”

Protein-flexible chain polymer interactions to explain protein partition in aqueous two-phase systems and the protein–polyelectrolyte complex formation

“…PEO forms aqueous two-phase systems, therefore liquid-liquid extraction can be used [20], while PVS and PAA have been used in the precipitation of proteins [21] by formation of non-soluble polyelectrolyte-protein complexes, being a coulombic mechanism of interaction.…”

Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques

“…The pH of each one was varied 0.50 units using HCl or NaOH solutions and for each value of pH the absorbance at 420 nm was measured at 25 • C. Then, the phase diagram of the complex was obtained by plotting absorbance at 420 nm vs pH [16].…”