Emission Quenching Mechanisms in Octopus vulgaris Hemocyanin: Steady-State and Time-Resolved Fluorescence Studies

Fluorescence emission properties of various derivatives of Octopus vulgaris hemocyanin, namely, oxy, deoxy, met, half-met, half-apo, and apo derivatives, are studied by means of time-resolved and quenching techniques. Fluorescence decay can be satisfactorily fitted by two-exponential analysis in all hemocyanin derivatives. Fluorescence quenching experiments, using acrylamide, iodide, and a combination of the two, are carried out in order to correlate the observed lifetimes with different classes of fluorophores, distinguishable by their accessibility to the external quenchers. Fluorescence lifetimes of 1.2, 2.1, and 5.5 ns are attributed to buried, partially exposed, and fully exposed tryptophans, respectively, in 11S hemocyanin at pH 8.5. For 50S hemocyanin, the lifetime pattern is very similar, but a shortening of all lifetime values is observed. The fluorescence of the class of partially exposed tryptophans, situated in close proximity to the active site, is totally quenched in oxy-...