ABSTRACT
Interactions between a model protein (bovine serum albumin--BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan-BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.
Subject(s)
Chemical Precipitation , Chitosan , Models, Chemical , Multiprotein Complexes/chemistry , Serum Albumin, Bovine , Animals , Cattle , Chitosan/chemistry , Chitosan/metabolism , Circular Dichroism , Hydrogen-Ion Concentration , Multiprotein Complexes/metabolism , Nephelometry and Turbidimetry , Protein Folding , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism , Solubility , Spectrometry, Fluorescence , TemperatureABSTRACT
Interactions between catalase and the cationic polyelectrolytes: chitosan and Eudragit E100 have been investigated owing to their scientific and technological importance. These interactions have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It was found that the catalase conformation does not change significantly during the chain entanglements between the protein and the polyelectrolytes. The effects of pH, ionic strength and anions which modify the water structure were evaluated on the polymer-protein complex formation. A net coulombic interaction force between them was found since the insoluble complex formation decreased after the NaCl addition. Both polymers were found to precipitate around 80% of the protein in solution. No modification of the tertiary and secondary protein structure or the enzymatic activity was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan and Eudragit E100 proved to be a useful framework to isolate catalase or proteins with a slightly acid isoelectrical pH by means of precipitation.
Subject(s)
Acrylates/chemistry , Catalase/chemistry , Chitosan/chemistry , Polyamines/chemistry , Polymers/chemistry , Acrylates/metabolism , Animals , Catalase/isolation & purification , Catalase/metabolism , Cattle , Chemical Precipitation , Chitosan/metabolism , Circular Dichroism , Enzyme Stability , Humans , Hydrogen-Ion Concentration , Nephelometry and Turbidimetry , Polyamines/metabolism , Polyelectrolytes , Polymers/metabolism , Protein Binding , Solubility , Spectrometry, Fluorescence , Temperature , TitrimetryABSTRACT
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.
Subject(s)
Chymotrypsin/metabolism , Light , Polyvinyls/metabolism , Scattering, Radiation , Sulfonic Acids/metabolism , Animals , Cattle , Chemical Precipitation , Diffusion/drug effects , Diffusion/radiation effects , Electrolytes , Kinetics , Nephelometry and Turbidimetry , Particle Size , Sodium Chloride/pharmacology , Solubility/drug effects , Solubility/radiation effects , Solutions , Temperature , TitrimetryABSTRACT
The influence of the phase volume ratio and polymer pausidispersity on chymosin and pepsin partition in polyethylenglycol-phosphate aqueous two-phase systems was studied. Both proteins showed a high affinity for the polyethylenglycol rich phase with a partition coefficient from 20 to 100 for chymosin and from 20 to 180 for pepsin, when the polyethyleneglycol molecular mass in the system varied between 1450 and 8000. The partition coefficient of chymosin was not affected by the volume phase ratio, while the pepsin coefficient showed a significant decrease in its partition coefficient with the increase in the top/bottom phase volume ratio.
