title: Fluorescence spectroscopy of the tryptophan microenvironment in Carcinus aestuarii hemocyanin.
abstract: The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues.
authors.affiliation: Department of Biology, University of Padova, Padova, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Di Muro
authors.forename: Mariano
authors.initials: M
authors.lastname: Beltramini
authors.forename: Peter
authors.initials: P
authors.lastname: Nikolov
authors.forename: Irina
authors.initials: I
authors.lastname: Petkova
authors.forename: Benedetto
authors.initials: B
authors.lastname: Salvato
authors.forename: Fernanda
authors.initials: F
authors.lastname: Ricchelli
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D003386:Brachyura; D006433:Hemocyanins / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis*
chemical_list: D001059:Apoproteins; D014364:Tryptophan; D006433:Hemocyanins