Multi-block poloxamer surfactants suppress aggregation of denatured proteins

https://doi.org/10.1016/j.bbagen.2007.08.017Get rights and content

Abstract

On the basis of elastic light scattering, we have compared the capacity of the multi-block, surfactant copolymers Poloxamer 108 (P108), Poloxamer 188 (P188), and Tetronic 1107 (T1107), of average molecular weight 4700, 8400, and 15,000, respectively, with that of polyethylene glycol (PEG, molecular weight 8000) to suppress aggregation of heat-denatured hen egg white lysozyme (HEWL) and bovine serum albumin (BSA). We also compared the capacity of P188 to that of PEG to suppress aggregation of carboxypeptidase A denatured in the presence of trifluoroethanol and to facilitate recovery of catalytic activity. In contrast to the multi-block copolymers, PEG had no effect in inhibiting aggregation of HEWL or of carboxypeptidase A with the recovery of catalytic activity. At very high polymer:protein ratios (≥ 10:1), PEG increased aggregation of heat-denatured HEWL and BSA, consistent with its known properties to promote macromolecular crowding and crystallization of proteins. At a polymer:protein ratio of 2:1, the tetra-block copolymer T1107 was the most effective of the three surfactant copolymers, completely suppressing aggregation of heat-denatured HEWL. At a T1107:BSA ratio of 10:1, the poloxamer suppressed aggregation of heat-denatured BSA by 50% compared to that observed in the absence of the polymer. We showed that the extent of suppression of aggregation of heat-denatured proteins by multi-block surfactant copolymers is dependent on the size of the protein and the copolymer:protein molar ratio. We also concluded that at least one of the tertiary nitrogens in the ethylene-1,2-diamine structural core of the T1107 copolymer is protonated, and that this electrostatic factor underlies its capacity to suppress aggregation of denatured proteins more effectively than nonionic, multi-block poloxamers. These results indicate that amphiphilic, surfactant, multi-block copolymers are efficient as additives to suppress aggregation and to facilitate refolding of denatured proteins in solution. Because of these properties, multi-block, surfactant copolymers are suitable for application to a variety of biotechnological and biomedical problems in which refolding of denatured or misfolded proteins and suppression of aggregation are important objectives.

Introduction

Refolding of proteins in non-native states and suppression of aggregation are important processes that occur under physiological conditions in vivo or that can be achieved through intervention in vitro with synthetic additives. A unifying principle common to both situations facilitating the recovery of biological function of proteins is mutual interaction of hydrophobic surfaces: In vivo the solvent exposed, hydrophobic residues of misfolded or denatured proteins interact with the surface of the inner cavity of chaperones [1], [2]. In experimental systems in vitro, we have shown in preliminary studies that the exposed hydrophobic residues of denatured proteins can interact with the surface of hydrophobic parts of synthetic additives introduced to facilitate refolding [3]. While the presence of multi-component chaperones in the cytosol is sufficient to prevent accumulation of aggregates of misfolded proteins under ordinary conditions, abrupt accumulation of aggregates of proteins can occur through pathophysiological processes underlying disease [4], [5] or, for instance, through thermal, electrical, or physical trauma requiring medical attention [6], [7], [8]. An important objective in our laboratory has been to develop use of multi-block, surfactant copolymers as synthetic agents administered through intravenous transfusion that can facilitate recovery from massive cell injury. A critical requirement of such polymers is the capacity to facilitate restoration of membrane function and to facilitate recovery of biological function and of the native structure of denatured proteins.

The chemical bonding structures of multi-block copolymers are schematically illustrated in Fig. 1. The surfactant copolymers known as Poloxamer 108 and Poloxamer 188 possess a tri-block structure comprised of two hydrophilic, polyoxyethylene (EO) segments and one central, hydrophobic, polyoxypropylene (PO) segment joined through ether oxygen linkages in an a:b:a construct. They are of average molecular weight 4700 and 8400, respectively. In contrast, the tetra-block copolymer Tetronic 1107, of average molecular weight 15,000, possesses a central ethylene-1,2-diamine skeleton to which the polyoxypropylene segments are joined to form a hydrophobic core with terminal, hydrophilic, polyoxyethylene segments flanking the hydrophobic core [9]. The T1107 copolymer is, thus, seen to contain two tertiary, organic nitrogens buried within the central hydrophobic core unlike the non-ionic P108 and P188 copolymers.

Of the tri-block surfactants, P188 has been used more extensively than P108 in pharmacological and physiological studies [10], [11], [12], [13], [14]. We have previously demonstrated that P188 significantly improves survival of cells following injury in which loss of membrane integrity occurs [6], [7], [10]. The results of these investigations, thus, demonstrate that the P188 copolymer is associated with a significant capacity to facilitate repair of damaged membranes. In addition, administration of P188 has been shown to mitigate the duration and pain associated with sickle-cell crises [15]. In contrast to multi-block poloxamer surfactants, PEG is comprised only of a hydrophilic polyoxyethylene chain and is non-ionic. Nonetheless, PEG, available as a commercial product over a broad range of molecular weight (200–500,000), has been employed to facilitate refolding of denatured proteins and to suppress their aggregation [16], [17], [18] and to restore nerve cell function by facilitating repair of damaged cell membranes [19], [20], [21]. The strikingly different character of poloxamer surfactants comprised of hydrophobic and hydrophilic segments from that of PEG suggests that they promote refolding of proteins and repair of cellular membranes through different mechanisms.

In a preliminary investigation, we compared the capacity of P188 and PEG (average molecular weight 8000) to facilitate recovery of the catalytic activity of heat-denatured HEWL [3]. We observed that the surfactant poloxamer restored catalytic action of the enzyme to a significant extent while PEG had no effect. Because our results with respect to PEG appeared to contradict the observations of others with respect to aggregation and refolding of denatured proteins [16], [17], [18], [22], [23], [24], we have investigated these differences in greater detail. On the basis of elastic light scattering measurements, we have compared the capacity of P108, P188, and T1107 to that of PEG to suppress aggregation of thermally denatured HEWL and BSA. We have also compared the capacity of P188 to that of PEG to promote recovery of catalytic activity of chemically denatured carboxypeptidase A (CPA).

Because the polymers employed in this study differ in molecular weight, they differ from each other not only with respect to hydrodynamic volume and dimensions but also with respect to their size relative to each protein. Accordingly, we have paid close attention to the stoichiometry of added polymer to each protein in solution. PEG showed essentially no capacity to retard aggregation of the denatured proteins and did not restore catalytic function of denatured CPA. As the polymer:protein ratio was increased, light scattering results showed that PEG promoted only an increase in the molecular weight and concentration of protein aggregates, consistent with its characteristic properties of macromolecular crowding [25], [26], [27]. On the other hand, the capacity of the surfactant poloxamers to reduce accumulation of protein aggregates was significant and followed the order T1107 > P188 > P108.

Section snippets

Materials

Lyophilized HEWL, fatty-acid free BSA, and bovine pancreatic CPA (Cox preparation) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The surfactant copolymers P108, P188, and T1107 were obtained from BASF Corporation (Mt. Olive, NJ, USA) and used as received. The mean molecular weights were 4700, 8400, and 15,000 for P108, P188, and T1107, respectively. PEG (average molecular weight 8000) and TFE were obtained from Sigma-Aldrich (Milwaukee, WI, USA). HEWL was dissolved in 150 mM KCl

Lysozyme

Fig. 2 compares the Rayleigh light scattering intensity of solutions of HEWL prior to and after heating to 90 °C for 30 min followed by temperature re-equilibration to 25 °C. While the light scattering intensity for HEWL solutions did not vary significantly with protein concentration prior to heating, it is seen that the intensity increased significantly after heating and re-equilibration to 25 °C. Compared to the increase in light scattering intensity observed for HEWL solutions with no added

Discussion

The importance of classical elastic light scattering, known as Rayleigh scattering, from particles in solution of dimension no greater than λ/20 (where λ is the wavelength of incident light) is that the intensity of the scattered radiation is proportional to the molecular weight and concentration of the solute particle [30]. Upon denaturation, the molecular dimensions of the globular proteins employed in this investigation do not change significantly with respect to the wavelength of incident

Conclusions

Macromolecules, such as PEG, facilitate refolding of denatured proteins through an excluded volume effect generally called macromolecular crowding. However, under conditions of high protein concentrations, there is little accumulation of refolded proteins because the presence of such “inert background macromolecules” leads to greatly increased rates of association and aggregation of denatured proteins without recovery of function. Treatment of massive cell injury, as can occur in instances of

Acknowledgments

This work was supported by grants from the National Institutes of Health to R.C.L. (GM 64757 and R01 GM61101) and in part by the Department of Biochemistry and Molecular Biology. C.M.S. was a 2005 Ronald E. McNair Summer Undergraduate Research Fellow at the University of Chicago.

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