Darío Spelzini

Chymotrypsin-poly vinyl sulfonate interaction studied by dynamic light scattering and turbidimetric approaches

The formation of non-soluble complexes between a positively charged protein and a strong anionic polyelectrolyte, chymotrypsin, and poly vinyl sulfonate, respectively, was studied under different experimental conditions such as pH (1-3.5), protein concentration, temperature, ionic strength, and the presence of anions that modifies the water structure. Turbidimetric titration and dynamic light scattering approaches were used as study methods. When low protein-polyelectrolyte ratio was used, the formation of a soluble complex was observed. The increase in poly vinyl sulfonate concentration produced the interaction between the soluble complex particules, thus inducing macro-aggregate formation and precipitation. Stoichiometry ratios of 500 to 780 protein molecules were found in the precipitate per polyelectrolyte molecule when the medium pH varied from 1.0 to 3.5. The kinetic of the aggregation process showed to be of first order with a low activation energy value of 4.2+/-0.2 kcal/mol. Electrostatic forces were found in the primary formation of the soluble complex, while the formation of the insoluble macro aggregate was a process driven by the disorder of the ordered water around the hydrophobic chain of the polymer.

Polyethyleneglycol–pepsin interaction and its relationship with protein partitioning in aqueous two-phase systems

The interaction between the acidic protein, pepsin, and the non-charged polyethyleneglycol polymer was studied by dynamic light scattering, fluorescence spectroscopy and measurements of the protein thermal stability at neutral pH. Polyethyleneglycol of average molecular mass 1450 showed a higher interaction capacity with the protein than polyethyleneglycol of average molecular mass 8000. Polyethyleneglycol of average molecular mass 1450 showed a molecular mechanism where the interpolymer interaction led to the complex formation. This fact can be explained taking into account that the extended form on this polymer molecule favours the interaction with the protein, which is highly dependent of the polymer total concentration. Polyethyleneglycol of average molecular mass 8000 showed a cooperative interaction between the polymer and protein molecules which was independent of the PEG concentration.

Pepsin extraction from bovine stomach using aqueous two-phase systems : Molecular mechanism and influence of homogenate mass and phase volume ratio

Pepsin partitioning, a gastric acid protease, in aqueous two-phase systems of polyethyleneglycol/potassium phosphate, sodium citrate and ammonium sulphate was assayed using polyethylenglycol of different molecular mass. Pepsin was found to be partitioned towards the polymer-rich phase in all the systems, which suggests an important protein-polymer interaction due to the highly hydrophobic character of the protein surface exposed to the solvent. The pepsin partitioning behavior was explained according to Timasheffs preferential interaction theory. The process was driven entropically with participation of structured water around the polyethyleneglycol ethylenic chains. The best pepsin recovery was observed in the systems polyethyleneglycol molecular mass 600. These systems were chosen in order to assay the bovine stomach homogenate partition and to compare different working conditions such as the top-bottom phase volume ratio and homogenate proportions in the total system. The best purification factors were obtained with PEG600/potassium phosphate with low top-bottom volume ratio using 15% of bovine stomach homogenate in the system total mass.