Renato E.F. Boto

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. dynamic binding capacity using lysozyme as a model

A constant development of dye-affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye-affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30 degrees C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K(d)) obtained was 2.61 +/- 0.36 x 10(-5 )m, proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme.

A study of N,N′-dicarboxyalkylthiacarbocyanines as cyanine direactive dyes covalently bound to cellulose

Abstract The synthesis of N,N ′-dicarboxyalkylthiacarbocyanines and their spectroscopic properties before and fixation to microcrystalline cellulose are described. Fixation yields were determined by FTIR spectroscopy, giving an alternative to the classical indirect spectroscopic method that uses the ratio of the areas of the absorption bands for cellulose and fixed dye. Diffuse reflectance spectrometry was used to compare chemically bound dye on cellulose to physically adsorbed dye. These results, in tandem with laser induced fluorescence emission data, revealed the formation of aggregates and considerable molecular distortion, the latter resulting from a decrease in molar absorptivity compared to the case in which the dye is physically absorbed.

Study of specific interaction between nucleotides and dye support by nuclear magnetic resonance

The binding between four matrices (beaded cellulose, cellulose acetate, cellulose triacetate and Sepharose CL‐6B) and beaded cellulose derivatized with a thiacarbocyanine dye with 5′‐mononucleotides is investigated by Saturation Transfer Difference Nuclear Magnetic Resonance (STD–NMR) technique. This procedure intends to identify unspecific interactions between 5′‐mononucleotides and matrices commonly used in affinity chromatography systems and also clarify the contribution of a thiacarbocyanine dye immobilized onto cellulose beads in a biorecognition process. The differences between non‐derivatized and derivatized beaded cellulose evidence the contribution of thiacarbocyanine dye in the observed interaction. STD–NMR experiments show that Sepharose CL‐ 6B interact less with the 5′‐mononucleotides comparatively with beaded cellulose. Indeed, beaded cellulose shows nonspecific interactions with almost all 5′‐mononucleotides that compromises the specificity of the interaction between the thiacarbocyanine dye immobilized with the 5′‐mononucleotides. The cellulose matrices where the hydroxyl groups are replaced by acetate and triacetate groups do not exhibit binding response to the 5′‐ mononucleotides, whereas the thiacarbocyanine dye contribution is evidenced by the reinforcement of the interactions with the sugar moiety of 5′‐GMP and 5′‐UMP and with base of 5′‐AMP, 5′‐CMP and 5′‐TMP. This screening of the nucleotide atoms involved in the binding to the supports can be very useful in chromatography evaluations in which dye‐affinity chromatography supports may be used, such as purification of nucleic acids. Copyright

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

New unsymmetrical aminosquarylium cyanine dyes were synthesized and their potential as photosensitizers evaluated. New dyes, derived from benzothiazole and quinoline, were prepared by nucleophilic substitution of the corresponding O-methylated, the key intermediate that was obtained by methylation with CF3SO3CH3 of the related zwitterionic unsymmetrical dye, with ammonia and methylamine, respectively. All three news dyes herein described displayed intense and narrow bands in the Vis/NIR region (693-714nm) and their singlet oxygen formation quantum yields ranged from 0.03 to 0.05. In vitro toxicity, in Caco-2 and HepG2 cells, indicated that dark toxicity was absent for concentrations up to 5µM (for the less active dye) or up to 1µM (for the two more active dyes). The three dyes present potential as photosensitizers, differing in irradiation conditions and period of incubation in the presence of irradiated dye. The less active dye needs a longer irradiation period to exhibit phototoxicity which is only evident after longer period of contact with cells (24h). However, the remaining two more active dyes produce higher phototoxicity, even at shorter incubation periods (1h), with shorter irradiation time (7min). Although in different extents, these dyes show promising in vitro results as photosensitizers.

NMR screening of new carbocyanine dyes as ligands for affinity chromatography

Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N-carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α-chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un-substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α-chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α-chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD-NMR technique was successfully used to screen cyanine-protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography.