Proximity Relationships of Tryptophanyl Residues and Oxygen Binding Site in Levantina hierosolima Hemocyanin. A Fluorimetric Study

Nurith Shaklai, Ari Gafni, Ezra Daniel*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The effect of iodide on the fluorescence of Levantina hierosolima hemocyanin was studied. About half of the tryptophanyl fluorescence yield of the apoprotein was found to be accessible to quenching by iodide. In contrast, no quenching of the fluorescence of oxyhemocyanin by iodide could be detected. It was concluded that the residual fluorescence of oxyhemocyanin emanates exclusively from buried tryptophans. A comparison of the polarization of the fluorescence of deoxy- and oxyhemocyanin, as well as of apohemocyanin in the absence and presence of iodide, does not provide evidence for significant tryptophan-tryptophan energy transfer. The fluorescence decay of apohemocyanin could be fitted to a biexponential decay function with lifetimes of 4.6 and 1.2 ns, with amplitudes 0.30 and 0.70, respectively. The fluorescence decay of deoxyhemocyanin was virtually identical to that of the apoprotein, showing that the introduction of copper did not affect the emissive properties of the protein. Analysis of the decay curve of oxyhemocyanin showed two lifetimes, 3.8 and 0.71 ns with amplitudes of 0.17 and 0.83, respectively. Comparison of the quantum yields calculated from the decay kinetics with the ones measured directly showed that 0.55 of the tryptophanyl emission in deoxyhemocyanin was totally quenched by the introduction of oxygen. Our findings indicate that the copper-binding site in hemocyanin is located by the solvent-accessible tryptophans, near the exterior of the molecule.

Original languageEnglish
Pages (from-to)4438-4442
Number of pages5
JournalBiochemistry
Volume17
Issue number21
DOIs
StatePublished - 1978

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