P. Di Muro

THE O-DIPHENOL OXIDASE ACTIVITY OF ARTHROPOD HEMOCYANIN

Arthropod hemocyanin (isolated from the crab Carcinus maenas and the lobster Homarus americanus) is usually referred to as an oxygen transport‐storage protein. The protein, however, also catalyses with low efficiency the oxidation of o‐diphenol to quinone, similarly to tyrosinase (monophenol,o‐diphenol: oxygen oxidoreductase). The enzymatic parameters of hemocyanin are affected by the aggregation state of the protein; namely V max exhibited by a dissociated subunit is one order of magnitude greater than that of aggregated species. The reaction velocity is increased by the presence of perchlorate, an anion of the Hofmeister series. The results are also discussed on the basis of active site accessibility in comparison with tyrosinase.

Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin.

The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly alpha-helical domain that directly binds the copper ions of the reaction center and a beta-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol.

Saccharose solid matrix embedded proteins: a new method for sample preparation for X-ray absorption spectroscopy.

Abstract In this study, solid samples of hemoglobin and hemocyanin have been prepared by embedding the proteins into a saccharose-based matrix. These materials have been developed specifically for specimens for X-ray absorption spectroscopy (XAS). The preservation of protein conformation and active site organization was tested, making comparisons between the solid and the corresponding liquid samples, using resonance Raman, infra red, fluorescence and XAS. The XAS spectra of irradiated solid and liquid samples were then compared, and the preservation of biological activity of the proteins during both preparation procedure and X-ray irradiation was assessed. In all cases, the measurements clearly demonstrate that protein solid samples are both structurally and functionally quite well preserved, much better than those in the liquid state. The saccharose matrix provides an excellent protection against X-ray damages, allowing for longer exposure to the X-ray beam. Moreover, the demonstrated long-term stability of samples permits their preparation and storage in optimal conditions, allowing for the repetition of data collection with the same sample in several experimental sessions. The very high protein concentration that can be reached results in a significantly better signal-to-noise ratio, particularly useful for high molecular weight proteins with a low metal-to-protein ratio. On the bases of the above-mentioned results, we propose the new method as a standard procedure for the preparation of biological samples to be used for XAS spectroscopy.

Functional SAXS study of haemocyanin dioxygen-carrier protein

Abstract The effects of conformational rearrangements on dioxygen binding to molluscan haemocyanins have been investigated by small-angle X-ray scattering (SAXS). The SAXS patterns of the oxygenated and deoxygenated forms of Octopus vulgaris haemocyanin are significantly different; whereas the patterns of the two forms of Rapana thomasiana haemocyanin are almost superimposable. A program has been developed, based on the differences in molecular dimensions, in order to simulate the effects observed in the investigation.

The use of small-angle X-ray scattering in the study of quaternary organisation of giant proteins

Abstract We report an investigation on the quaternary organisation of hemocyanin giant proteins, performed with continuous small-angle X-ray scattering (SAXS) at the LURE facility. The molecular weight of a 11S subunit of molluscan hemocyanin was determined from the experimental data. Structural models of this subunit, which include seven or five functional units, were also derived and correlated to the whole molecule, which has the form of a hollow cylinder.