Vânia C. Graça

Protein purification by aminosquarylium cyanine dye-affinity chromatography

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.

Ethylenediamine-Derived Chromatographic Ligand to Separate BSA, Lysozyme, and RNase A

Chromatography has become an essential tool for the purification of proteins, since most purification schemes involve some forms of this methodology. Recently, using chromatographic matrices prepared from symmetrical aminosquarylium cyanine dyes immobilized on Sepharose via a central alkylamino residue, we were able to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. Following this, we envisioned that the immobilization of an asymmetric squarylium dye bearing an N-carboxyethyl group in one of its ending nuclei, on ethylenediamine-activated Sepharose, through EDC/NHS amidation coupling, could enhance the ligand’s mobility and improve the interactions with the target proteins. The prepared support was found to separate an artificial mixture of BSA, lysozyme, and RNase A. Unexpectedly, the support prepared in the absence of the dye exhibited a separation performance similar to that of the dyed support, contrary to that observed in all previous studies using cyanine dyes as ligands for affinity chromatography, which prompted us to try to determine the structural molecular constitution of the matrix surface. A synthetic route to the final chromatographic support could be devised, which is believed to consist in the cyclization of two nearby ethylenediamine units, involving the inclusion of a succinimide-derived residue between them and the EDC-mediated Lossen rearrangement of an intermediary hydroxamic acid.

Aminosquaraines as potential photodynamic agents: Synthesis and evaluation of in vitro cytotoxicity

The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-called phototherapeutic window (650-850nm) is described. Their cytotoxicity, under dark and illuminated conditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearly exhibiting photodynamic activity. The photosensitizing ability against some cell lines, together with the low toxicity for the non-tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into potential candidates as photosensitizers for PDT.

Protein purification by aminosquarylium cyanine dye-affinity chromatography.

Affinity chromatography (AC) is one of the most important techniques for the separation and purification of biomolecules, being probably the most selective technique for protein purification. It is based on unique specific reversible interactions between the target molecule and a ligand. In this affinity interaction, the choice of the ligand is extremely important for the success of the purification protocol. The growing interest in AC has motivated an intense research effort toward the development of materials able to overcome the disadvantages of conventional natural ligands, namely their high cost and chemical and biological lability. In this context, synthetic dyes have emerged, in recent decades, as a promising alternative to biological ligands. Herein, detailed protocols for the assembling of a new chromatographic dye-ligand affinity support bearing an immobilized aminosquarylium cyanine dye on an agarose-based matrix (Sepharose CL-6B) and for the separation of a mixture o f three standard proteins: lysozyme, α-chymotrypsin, and trypsin are provided.