sepharose and alpha-chymotrypsin

The interest for a better understanding of ion-exchange mechanisms at the atomic level has strongly increased over the past decades. Indeed, molecular-level information about physico-chemical mechanisms could help optimizing chromatographic processes for protein purification, which are sub-optimized due to the lack of predictive models. A promising approach is based on the use of Molecular Dynamics (MD) simulations to study local phenomena inside adsorbents which can then be challenged against experimental results. In this work, macroscopic experimental data, consisting in the ion-exchange uptake of α-chymotrypsin onto SP Sepharose FF, have been compared to the adsorption behavior predicted by MD simulations. The chromatographic surface, represented as a uniform distribution of ligands with a counterion layer, in the presence of the protein was modeled using all-atom representation. The SMA formalism was used to describe single adsorption isotherms and to relate macroscopic observations with molecular simulations. Two SMA parameters based on physical principles, the characteristic charge n and the steric factor σ, have been estimated by both experiments and MD simulations. At pH 5 and NaCl concentration of 100 mM, our study shows a fairly good agreement between both results, especially for the characteristic charge. It is shown that the steric factor calculation is strongly dependent on the ligand density on the adsorbent surface, whose value must be carefully determined in order to obtain reliable predictions. In addition, four binding patches were identified as being involved in the adsorption, which have been confirmed through binding free energy calculations.

Topics: Adsorption; Cation Exchange Resins; Chromatography, Ion Exchange; Chymotrypsin; Ligands; Molecular Dynamics Simulation; Sepharose

The synthesis of the dipeptide N-benzoyl-l-tyrosine-l-argininamide (BTAA) was conducted under kinetic control with N-benzoyl-l-tyrosine ethyl ester as acyl donor and argininamide as nucleophile using immobilized α-chymotrypsin as catalyst. Using a mathematical procedure, the kinetic constants corresponding to the proposed mechanism of peptide synthesis were determined in three different cosolvent media, namely, ethanol, diglyme and acetonitrile. These constants were used for evaluating the selectivity of glyoxyl-agarose immobilized α-chymotrypsin in the synthesis of BTAA by determining the ratios of synthesis to hydrolysis rates.

Topics: Animals; Arginine; Biocatalysis; Cattle; Chymotrypsin; Dipeptides; Enzymes, Immobilized; Glyoxylates; Hydrolysis; Kinetics; Sepharose; Solvents; Tyrosine

Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 μmol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼ 60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, β-lactoglobulin and α-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.

Topics: Adsorption; Chromatography; Chymotrypsin; Dendrimers; Hydrophobic and Hydrophilic Interactions; Lactoglobulins; Ligands; Proteome; Sepharose

Displacer lead compounds were selected from a commercially available database to identify potential selective displacers for a binary protein mixture in ion exchange chromatography. Parallel batch screening experiments were carried out with these lead compounds to study the effect of displacer concentration on the relative amounts of the proteins displaced. Experiments were conducted with a mixture containing ribonuclease A and alpha-chymo-trypsinogen A which exhibited very similar retention behavior under linear gradient conditions. The batch displacement results indicated that most of these lead compounds were indeed selective for displacing ribonuclease A. In fact, one of these displacers exhibited extremely high selectivity, displacing essentially all of the ribonuclease A while displacing minimal alpha-chymotrypsinogen A at a displacer concentration of 10 mM. These results were validated under column conditions, with the ribonuclease A being displaced and the alpha-chymotrypsinogen A remaining on the column after the displacer breakthrough. In order to examine whether this was mass action or chemically selective displacement, an affinity ranking plot based on the Steric Mass Action (SMA) model was generated, and the results confirmed that this was not a mass action displacement. In order to test the hypothesis that displacer protein binding was playing a role in these separations, Surface Plasmon Resonance (SPR) was carried out. The results suggest that while the chemically selective displacer interacted with alpha-chymo-trypsinogen A, it had no interaction with ribonuclease A. The ability to exploit protein displacer binding in concert with appropriate displacer resin affinities opens up new possibilities for creating selective displacement systems.

Topics: Binding, Competitive; Chromatography, Affinity; Chromatography, Ion Exchange; Chymotrypsin; Enzymes, Immobilized; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Chemical; Protein Binding; Ribonuclease, Pancreatic; Sepharose; Structure-Activity Relationship; Surface Plasmon Resonance

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.

Topics: Animals; Catalysis; Cattle; Chymotrypsin; Electron Spin Resonance Spectroscopy; Enzyme Activation; Enzyme Stability; Enzymes, Immobilized; Indoles; Membranes, Artificial; Models, Chemical; Pancreas; Sensitivity and Specificity; Sepharose; Structure-Activity Relationship

The major portion of rat brain hexokinase (HK type 1) is bound to the outer membrane of mitochondria and glucose-6-phosphate (G6P) can release the bound enzyme. In an attempt to look at the 'hydrophobic' component of binding, interaction of the enzyme with a purely hydrophobic matrix, palmityl-substituted Sepharose-4B (Sepharose-lipid) was investigated. Hexokinase readily bound to this matrix with retention of its catalytic activity. Glucose-6-phosphate which has a releasing effect on the mitochondrially bound enzyme, enhanced binding of the enzyme on the hydrophobic matrix. Chymotrypsin treatment of hexokinase which causes loss of binding to mitochondria, also results in loss of adsorption to the hydrophobic matrix, thus demonstrating that the 'hydrophobic tail' present at its N-terminal end is essential for binding in both cases. Data presented provide some new information relevant to understanding how hexokinase interacts with its natural binding matrix, the mitochondrion.

Topics: Adsorption; Animals; Brain; Catalysis; Chromatography, Agarose; Chymotrypsin; Glucose-6-Phosphate; Hexokinase; Hydrophobic and Hydrophilic Interactions; Intracellular Membranes; Magnesium; Male; Mitochondria; Polyamines; Protein Binding; Protein Structure, Tertiary; Rats; Rats, Inbred Strains; Sepharose; Spermine