The binding of 1-anilino-8-naphthalene sulfonate to the hemocyanin of Octopus vulgaris.

The binding of 1-anilino-8-naphthalene sulfonate (ansyl) to native and copper-free hemocyanin of Octopus vulgaris has been studied in different conditions by measuring the fluorescence properties of the probe in the presence of hemocyanin. Native hemocyanin, either in the oxygenated or in the deoxygenated state, does not bind ansyl. The binding of ansyl with apohemocyanin induces a strong increase (from 0.004 to 0.6 -- 0.7) of the quantum yield and a blue shift from 520 nm to 460 nm of the emission maximum indicating the presence of ansyl binding sites in the protein. Experimental evidence is reported that the binding occurs at the copper-binding site of the protein. The dissociation constants of the ansyl-hemocyanin complexes are equal to about 10(-4) M, i.e. they are of the same order of those obtained with other proteins. The number of binding sites (n) of apohemocyanin for ansyl depends on the conformational state of the protein and ranges from 0.15 -- 0.80 mol/mol protein (Mr 50,000), depending on pH, ionic strength, and urea concentration. A negative interaction between the ansyl binding sites has been suggested.