title: Protein purification by aminosquarylium cyanine dye-affinity chromatography.
abstract: The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001, Covilhã, Portugal.
authors.forename: M S
authors.initials: MS
authors.lastname: Silva
authors.forename: V C
authors.initials: VC
authors.lastname: Graça
authors.forename: L V
authors.initials: LV
authors.lastname: Reis
authors.forename: P F
authors.initials: PF
authors.lastname: Santos
authors.forename: P
authors.initials: P
authors.lastname: Almeida
authors.forename: J A
authors.initials: JA
authors.lastname: Queiroz
authors.forename: F
authors.initials: F
authors.lastname: Sousa
mesh_terms: D002232:Carbocyanines / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D008024:Ligands; D011506:Proteins
keywords: affinity chromatography; aminosquarylium cyanine dyes; lysozyme; trypsin; α-chymotrypsin