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 Ca 2+ 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. Zn 2+ , when added to the protein, induced only a small perturbation and the emission maximum shifted to 481 ± 2 nm. Ca 2+ was able to perturb the fluorophore in the presence of Zn 2+ . 2‐ p ‐Toluidinylnaphthalene‐6‐sulfonate (TNS)‐labelled α‐subunit when excited at 345 nm exhibited very little fluorescence in the absence of Ca 2+ . Addition of Ca 2+ 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 Ca 2+ moves to a hydrophobic domain. The fact that Ca 2+ and Zn 2+ 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.