INVESTIGADORES
TUBIO Gisela
congresos y reuniones científicas
Título:
Experimental determination and thermodynamic modelling of biomolecule partitioning in aqueous two-phase systems containing biodegradable salts.
Autor/es:
PÉREZ, BRENDA; PELEGRINI MALPIEDI, LUCIANA; TUBIO, GISELA; NERLI, BIBIANA; PESSÔA FILHO, PEDRO DE ALCÂNTARA
Lugar:
Puerto Vallarta
Reunión:
Congreso; 16th International Conference on Biopartitioning and Purification BPP 2011; 2011
Resumen:
Aqueous two phase systems (ATPSs) formed by a polymer and a lyotropic sal
provide a powerful method for separating biomaterials. Those ATPSs containing
biodegradable anions such as citrate, tartrate and succinate are particularly
interesting due to the lower environmental impact. However, their use in
downstream processing has been hampered by the lack of a theory based on
fundamental principles capable of explaining and predicting the experimental
trends observed. The phase behavior of polymer/salt systems is complicated due
to both the size differences between the molecules to the nature of the mutual
interactions. Models for the excess Gibbs energy based in Pitzer equation have
been successfully employed to describe thermodynamic properties of pure
electrolyte solutions. They combine contributions from long and short-range
interactions.
In this work a modified Pitzer model was applied to solutions that
contain both an electrolyte and a neutral polymer in order to calculate partition
coefficients of biological molecules in ATPSs. The modifications accounts for
i) the dependency of model binary parameters -corresponding to polymer/polymer
and polymer/salt short range interactions- on the polymer chain length; ii)
the polymer presence like a second
solvent that affects density and dielectric constant on the long-range
interaction term.
ATPS formed by polyethyleneglycols of different MWs (4,000; 6,000 and
8,000) and sodium salts (citrate,
tartrate and succinate) were characterized. Polymer and salt concentrations in both
phases at the equilibrium condition were measured by independent analytical
methods. Partition coefficients (Kp) of seven model proteins: bovine serum
albumin, catalase, beta-lactoglobulin, alpha-amylase, lysozyme, pepsin, urease
and trypsin in the selected ATPSs were determined spectrophotometrically.
The first step in the calculation was to solve phase equilibrium in each
ATPS in the absence of partitioned biomolecule. Experimental liquid-liquid
equilibrium data were used to estimate the model binary parameters
corresponding to polymer/polymer, polymer/salt and cation/anion interactions through
the minimization of the deviation between experimental and calculated
compositions. Comparison between experimental and calculated tie line data
showed mean deviations always lower than 3 % thus indicating a good
correlation.
In the second step, partitioning of model proteins was analyzed. Pepsine
and lisozyme showed to be displaced to the polymer rich.phase (Kp>1) in all
the assayed systems while catalase and alpha amylase partitioned unilaterally to
the salt-rich phase (Kp <1). Different partitioning trends were observed for
the rest of assayed proteins. The model parameters corresponding to binary
interaction polymer-protein and salt-protein were then fitted to experimental
partition coefficients for each protein. The equilibrium compositions of both
phases calculated in absence of biomolecule were assumed to hold in its
presence due to the low protein concentration.
Predicted and experimental partition coefficients showed a quite
successful correlation. Mean deviations showed to be lower than the
experimental uncertainty for most of assayed proteins.
We conclude that the proposed modified Pitzer model provides a simple thermodynamic
framework for correlating and ultimately predicting phase separation and
protein partition coefficients. This could contribute to improve the design and
optimization of purification processes.The authors gratefully acknowledge financial support
from CAPES (Brasil) and MinCyT, PICT508/2006, PIP00196 (Argentina)