title: Study of catalase adsorption on two mixed-mode ligands and the mechanism involved therein.
abstract: Mixed-mode chromatography sorbents n-hexylamine HyperCel™ (HEA) and phenylpropylamine HyperCel™ (PPA) were evaluated for the study of adsorption of catalase from two different sources. Various parameters such as buffer composition, ionic strength and pH were investigated to study the mechanism of interaction of commercially available pre-purified catalase from Bovine liver, purified catalase from black gram (Vigna mungo) and crude extract of black gram containing catalase with these mixed-mode ligands. A simple and economical screening protocol for identifying optimal buffer conditions for adsorption and desorption of catalase was established with micro volumes of the sorbent in batch mode. With HEA HyperCel, it was observed that pre-purified catalase from both bovine liver and black gram was completely retained at pH 7.0, irrespective of the presence or absence of NaCl in the adsorption buffer, whereas the catalase from crude extract of black gram was completely retained only in the presence of 0.2 M salt in the adsorption buffer. The elution of catalase from both the sources was accomplished by lowering the pH to 4.5 in absence of salt. In case of PPA HyperCel, catalase from both the sources was very strongly adsorbed under different buffer conditions studied, and elution did not yield a significant catalase activity. From the screening experiments, it could be concluded that the interaction of catalase with HEA HyperCel could be dominated by hydrophobic forces with minor contributions from ionic interaction and with PPA HyperCel, it could be a combination of different non-covalent interactions acting on different loci on the surface of the protein.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore, India.
authors.forename: S
authors.initials: S
authors.lastname: Shiva Ranjini
authors.forename: M A
authors.initials: MA
authors.lastname: Vijayalakshmi
mesh_terms: D000327:Adsorption; D000818:Animals; D002021:Buffers; D002374:Catalase / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D027806:Lens Plant / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D009994:Osmolar Concentration; D010940:Plant Proteins / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D002021:Buffers; D010940:Plant Proteins; D012965:Sodium Chloride; D014508:Urea; D002374:Catalase