Fluorescence studies on the Ca2+and Zn2+binding properties of the α-subunit of bovine brain S-100a protein

Abstract

The single cysteine on the α-subunit of bovine brain S-100a protein has been modified with the thiol specific probe, Acrylodan. When the labelled apoprotein was excited at 380 nm the fluorescence emission maximum was centered at 484 ± 2 nm, suggesting that the probe is in a fairly hydrophobic environment. Addition of Ca²⁺ to the protein caused the emission maximum to undergo a red shift to 504 ± 2 nm, implying that the fluorophore is now more exposed to the solvent. Zn²⁺, when added to the protein, induced only a small perturbation and the emission maximum shifted to 481 ± 2 nm. Ca²⁺ was able to perturb the fluorophore in the presence of Zn²⁺. 2-p-Toluidinylnaphthalene-6-sulfonate (TNS)-labelled α-subunit when excited at 345 nm exhibited very little fluorescence in the absence of Ca²⁺. Addition of Ca²⁺ resulted in an increase in TNS fluorescence accompanied by a blue shift of the emission maximum to 445 ± 1 nm indicating that the probe in the presence of Ca²⁺ moves to a hydrophobic domain. The fact that Ca²⁺ and Zn²⁺ can perturb the labelled sulfhydryl group in the presence of each other clearly demonstrates that the binding sites for the two metal ions must be different on the α-subunit as well as on the S-100a protein.