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.

Synthesis, spectroscopic characterization and biological evaluation of unsymmetrical aminosquarylium cyanine dyes

“…The IR spectra of 9-12 do not show any evidence of carbonyl absorptions at approximately 1700 cm À1 , nor exhibit a strong absorption band in the region of 1600 cm À1 , as reported in the literature 30,31 for symmetrical squarylium and N-alkylaminosquarylium dyes possessing the indolenine moiety. The IR spectra for the unsymmetrical aminosquarylium dyes 11 and 12 show a weak band around 1630 cm À1 which is similar to that reported in the literature for symmetrical aminosquarylium derivatives from benzothiazole and quinoline, 24,28,32 indicating a strong bonding-delocalization within the four-membered ring system.…”

3,3′‐Diamino‐N‐methyldipropylamine as a versatile affinity ligand

“…The ligand density of the support was found to be 0.395 mmol per gram of support and was estimated from the percentage of nitrogen determined by elemental analysis. This value is slightly higher than the ligand density of other recently prepared affinity matrices using benzothiazole‐based squarylium dyes as ligands, what can be advantageous for the binding of biomolecules and the capacity of the support.…”

“…Although being one of the most popular class or organic dyes, cyanine dyes have been scarcely used as ligands. Recently, we reported the use of several symmetrical aminosquarylium cyanine dyes derived from benzothiazole as ligands for the separation of a mixture of lysozyme, α‐chymotrypsin, and trypsin . In this case, the dye was immobilized in Sepharose using diaminoalkyl groups as spacer arms.…”

Affinity chromatography: A review of trends and developments over the past 50 years

“…The low cost, ease of immobilization onto supports, and stability of dyeligands have made these binding agents popular in affinity separations, as demonstrated in Figure 6, and especially for large-scale processes [126,127]. Examples of proteins that have been purified through the use of dye-ligands are HSA, lysozyme, fucoidan, lactoferrin, αchymotrypsin, and IgG [126,127,[235][236][237][238][239][240][241][242][243][244].…”