The interaction of a naphthalene dye with apomyoglobin and apohemoglobin

1-Anilino-8-naphthalene sulfonate binds stoichiometrically to a specific site on apomyoglobin and apohemoglobin. One mole of ANS† is bound per mole of apoprotein with a dissociation constant of the order of 10⁻⁵M. Myoglobin and hemoglobin do not bind ANS. The addition of hemin displaces it from its complex with apomyoglobin, suggesting that ANS and heme bind at the same site. Its fluorescence changes markedly when it is bound to the apoprotein. The quantum yield is two-hundredfold higher than in water, while the emission peak is shifted from green to blue by 60 mμ. These changes are attributed to the essentially non-polar environment of the bound ANS. A similar dependence of quantum yield and emission maximum on the polarity of the environment is observed for solutions of ANS in various organic solvents. Fluorescence polarization and optical rotatory dispersion results indicate that the compactness and high helix content of myoglobin are retained in large part in the apomyoglobin-ANS complex. Highly efficient energy transfer is observed from the aromatic amino acids of the protein to the bound ANS. The use of anilinonaphthalene sulfonates as fluorescent probes of non-polar regions in proteins is discussed.