Summary.A blue carotenoid-protein complex (2rn~x 635 rim) was extracted and purified from the carapace of the crayfish Procambarus clarkii. The complex was further liberated from astaxanthin, its prostetic group, causing dissociation into apoprotein subunits. Reconstitution of the complex from the various sub-units (isolated by chromatofocusing) plus astaxanthin was attempted. Apoprotein-size pigments of rose-purple color (2~ 545 nm) were obtained. It was found that both monomers are required in order to reconstitute a blue complex fairly similar in structure ot the native one. However, the native conformation was not completely recovered, as indicated by some differences in the UV spectrum. Key words. Carotenoproteins; protein reconstitution; astaxanthin.Carotenoid pigments frequently occur in nature, especially in crustaceans, specifically bound to proteins, constituting the socalled carotenoid-protein complexes or carotenoproteins. The mechanism of interaction between protein and carotenoid that accounts for the change in color of the latter, from orange when it is free to blue when bound to the protein, is the main subject of investigation in most studies of carotenoproteins at present. The approach to the elucidation of such a mechanism can be made by means of reconstitution experiments. In a previous report 1, we described some structural requirements of the carotenoid for its binding to the protein. Now, the reconstitution of the complex is studied from the protein side. Materials and methods. The blue carotenoprotein from the carapace of the crayfish Procambarus clarkii was extracted using the method of Quarmby et al. 2 and purified as described in Gfirate et al. 6. Once the carotenoprotein had been purified, its prosthetic group astaxanthin was liberated from the protein by adding N, N-dimethyl-formamide (DMF); this resulted in the dissociation of the complex into subunits 3. The apoprotein subunits so obtained were separated by chromatofocusing in a column (25 x 1.0 cm) of Polybuffer Exchanger PBE TM 94 (Pharmacia) by the method of Zagalsky 3. Two protein fractions were collected; the first one eluted at pH 6.2 and the second at pH 5.7. These fractions were identified by 6 M urea polyacrylamide slab gel electrophoresis, performed by the method of Laemmli and Favre 5, and called P~ and P2 respectively, according to the nomenclature,reported by Gfi.rate et al. 6. Previously it has been shown ~ that keto groups at positions 4 and 4' and hydroxyl groups at positions 3 and 3', or at spatially analogue positions, as well as an unmodified polyenic chain, are required in the carotenoid structure for binding to the protein. Later, by using the chromatofocusing technique, we obtained a sufficient amount of isolated apoprotein subunits to undertake reconstitution experiments in order to study separately the interaction of the native carotenoid (astaxanthin) with each of the subunits and with both. Reconstitution was achieved by the Britton et al. procedure 4.The following combinations were tried: subunit PI plus a...
Summary.A blue carotenoprotein from the crayfish Procambarus clarkii was extracted and purified. This carotenoprotein contains the carotenoid astaxanthin as a prosthetic group. In the present work we have identified by reconstitution, after removing the native carotenoid, some characteristics of the carotenoids that could bind to the apoprotein. The carotenoid must have two oxo groups at positions 4, 4' and two hydroxyl groups at positions 3, 3' the hexagonal or pentagonal end structure being indifferent. It has been proved that changes in the polyene chain structure such as triple bonds destroy this binding capacity.
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