Elsevier

Journal of Chromatography B

Volume 857, Issue 1, 15 September 2007, Pages 25-31
Journal of Chromatography B

Interaction of lysozyme with negatively charged flexible chain polymers

https://doi.org/10.1016/j.jchromb.2007.06.025Get rights and content

Abstract

The complex formation between the basic protein lysozyme and anionic polyelectrolytes: poly acrylic acid and poly vinyl sulfonic acid was studied by turbidimetric and isothermal calorimetric titrations. The thermodynamic stability of the protein in the presence of these polymers was also studied by differential scanning calorimetry. The lysozyme–polymer complex was insoluble at pH lower than 6, with a stoichiometric ratio (polymer per protein mol) of 0.025–0.060 for lysozyme–poly vinyl sulfonic acid and around 0.003–0.001 for the lysozyme–poly acrylic acid. NaCl 0.1 M inhibited the complex precipitation in agreement with the proposed coulombic mechanism of complex formation. Enthalpic and entropic changes associated to the complex formation showed highly negative values in accordance with a coulombic interaction mechanism. The protein tertiary structure and its thermodynamic stability were not affected by the presence of polyelectrolyte.

Introduction

Production of proteins is a prime biotechnological application and includes upstream and, often more expensive, downstream processing steps to obtain the final product in the desired purified form. Bioseparation steps for recovery of final product can account for 50–80% of overall production costs [1]. Most purification technologies employ precipitation of proteins as one of the initial operations aimed at concentrating the product stream for further downstream steps. Precipitation by salts, organic solvents and non-ionic polymers are well known and simple techniques for protein concentration [2], [3]. Attempts are usually made to derive some degree of purification of target products in the precipitation step. The precipitation methods used, however, lack selectivity and thus limit their purification potential. Substantial advantage can be gained if the precipitation step is imparted with some degree of target specific selectivity.

There are several studies in which attention should be called to the formation of a flexible polymer chain (PCF)–protein complex [4], [5]. In addition, with the intention of developing novel drug delivery systems, block and graft polymers with proteins have also been studied in terms of the formation of polymer–protein complexes, although the authors did not pay much attention to the role of the polymer moieties in the complex formation.

Complex formation by proteins with water-soluble synthetic polymers is interesting from two points of view [6]:

  • -

    First concerns the way in which globular proteins interact with flexible chain macromolecules through electrostatic, hydrogen bonding and hydrophobic interactions, an understanding of which could provide a better explanation of the mechanisms of macromolecular interaction available in nature.

  • -

    Second concerns the extent to which biochemical activity is maintained in the resulting complexes, the answer to which is central to the molecular design of composite protein–polymer systems, such as immobilized enzymes, as well as to the process design for protein separation using water-soluble polymers.

Protein precipitation by formation of insoluble-protein complex is a potential technique used in the isolation and purification of protein [7], [8], [9]. To make the use of PCF an attractive means of protein separation, two additional problems should be solved. The first problem is the protein recovery from the PCF medium and regeneration of the polymer. One of the potential solutions to this problem is the use of reversibly soluble systems based on a better understanding of factors those affecting phase state and stability of PCF–protein complex formed.

The second problem is the selectivity of the polymer–protein interaction. This problem could be addressed by specific interactions introduced by affinity ligands coupled to the polymer, so-called macroligands, so that a reversible biospecific water-soluble complex is formed with the desired target protein.

Precipitation methods have the advantage that low concentration of polymer is needed to precipitate the protein, but in some cases the protein loses part of its biological activity [10]; so, it is necessary to know the molecular mechanism by which PCF in aqueous solution interacts with proteins. We have used spectroscopic and calorimetric techniques to obtain information about the molecular mechanism of interaction between a basic model protein: lysozyme and two negatively charged polyelectrolytes: poly acrylic acid and poly vinyl sulfonate with the goal of applying this information in the polyelectrolyte–protein complex formation as tool for protein separation.

Section snippets

Chemical

Egg white lysozyme (LYZ) was purchased from Sigma Chem. Co. (USA) and the polymers poly acrylic acid, sodium salt (PAA), 25% (w/w) sol. in water molecular average mass 240 kDa and poly vinyl sulfonic acid, sodium salt (PVS) 25% w/w. sol. in water, molecular average mass 170 kDa, were purchased from Aldrich and used without further purification. Buffers of different pH were prepared at concentration of 50 mM: phosphate buffer pH 5.5 and 7.0, and acetic acid/acetate buffer pH 3.1. They were adjusted

Lysozyme turbidimetric titration curves with the polymers

Fig. 1, Fig. 2 show typical titration curves of LYZ with PVS and PAA, respectively, from which, two important characteristics were observed:

  • (i)

    at low polymer–protein ratio absorbance increases linearly with an increase in the polymer total concentration and,

  • (ii)

    when the polymer concentration increases, there is a plateau which depends on the medium pH.

The free LYZ concentration remaining in the titration system was measured by absorbance at 280 nm of the supernatant solutions from which the insoluble

Discussion

Current methods of protein purification involve an extensive series of steps and processes that increase the cost of the final product. New techniques for large-scale protein separation are therefore of interest. One of these involves the addition of polyelectrolytes, leading to selective protein phase separation. Proteins interact strongly with both synthetic and natural polyelectrolytes. These interactions are modulated by variables such as pH and ionic strength, and may result in soluble

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