Ionic and Apolar Interactions of Cibacron Blue F3GA with Model Compounds and Proteins Application to Protein Purification

Abstract

Studies of the interactions of Cibacron Blue F3GA in aqueous solutions with organic solvents, salts, oligo-, and poly-peptides by visible absorption difference spectroscopy indicate that the dye-sodium chloride (or any other salt) interaction results in a difference spectrum that has a characteristic positive peak at 690 nm and negative double minima at 630 and 585 nm. Such a “salt-like” or “ionic” spectrum is also obtained with polycations such as spermine, spermidine, oligolysines, polylysine, polyarginine, and protamine. In a striking contrast, the difference spectrum of the dye in binary aqueous solvents containing dioxan or t-butyl alcohol at moderately high concentrations, measured against water, displays a positive peak and shoulder at 655 and 610 nm, respectively with a small negative contribution at wavelengths less than 550 nm. We assign this type of spectrum to a “nonpolar” interaction of the dye with the organic cosolvent molecules. Such spectral characteristics have been widely noticed in several studies involving Cibacron Blue and proteins, thereby providing a basis for a reasonable interpretation for the interaction of the dye with the proteins. An understanding of the forces involved in dye-protein interactions is essential for protein purification studies using the technique of dye-ligand chromatography. Complete separation of pepsin and chymosin present in calf rennet has been achieved using Blue agarose chromatography. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with the dye ligand. Differential surface hydrophobicity of the two enzymes may also play a key role in the separation.