Fluorescence properties of hemocyanin from tarantula ( Eurypelma californicum): A comparison between the whole molecule and isolated subunits

Abstract

The tryptophan (Trp) fluorescence properties of the 24-meric hemocyanin (Hc) from the tarantula Eurypelma californicum and its subunits a, d and e were studied and compared with respect to the influence of the two copper atoms at the oxygen-binding site and the status of oxygenation. Fluorescence quenching experiments (by using acrylamide and iodide as external quenchers) and lifetime determinations suggested the occurrence of different classes of Trp responsible for the emission in oxygenated, deoxygenated and copper-depleted protein. On the basis of the different content and distribution of Trp in the isolated subunits and their position in the primary structure, each class of fluorophores was assigned to defined Trp residues. In 24-meric Hc, Trp-292 is mostly responsible for the fluorescence emission in the protein-oxygenated state. After oxygen removal, the major contribution probably arises from Trp-346 while in copper-depleted Hc, about ∼ 70% of fluorescence is due to Trp-195 and Trp-197. All these Trp are conserved in the different subunit types and appear to be located in the interior of the protein matrix, with the exception of Trp-292, which is partially exposed to the solvent. The ratio between the fluorescence quantum yields of oxy-, deoxy- and apo-24-meric Hc is ∼ 1:10:15. The drastic fluorescence decrease in oxy-Hc is mostly due to static quenching by copper ions on that classes of fluorophors (Trp-195, 197 and 346) localized very close to the active site (within 11 Å). Each isolated subunit exhibits a peculiar fluorescence behavior, due to the heterogeneity of Trp distribution. In particular, the fluorescence properties of subunit d are dominated by Trp-386 which is present only in this subunit and is fully exposed on the protein surface.