Alberto M. Gárate

A blue carotenoprotein from the carapace of the crab, Carcinus maenas

1. 1. A blue carotenoprotein (λ max = 625 nm) containing astaxanthin as prosthetic group was extracted and purified from the carapace of the crab, Carcinus maenas. 2. 2. The extraction was difficult to achieve, and the blue product was not always obtained. 3. 3. The blue carotenoprotein had an approximate mol. wt of 365,000 (gel filtration). Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated only a single polypeptide of 38,200 daltons, suggesting that the blue protein is an octameric form. 4. 4. In the presence of denaturing agents such as heat, organic solvents and SDS, the blue complex readily dissociated to liberate the orange carotenoid astaxanthin and a colourless apoprotein. No other lipid or sugar was detected.

Purification and characterization of the blue carotenoprotein from the caparace of the crayfish Procambarus clarkii (Girard)

Abstract The isolation, purification and characterization of a blue carotenoprotein isolated from the carapace of the crayfish Procambarus clarkii (Girard) are reported. The molecular weight of the complex has been determined by polyacrylamide gradient gel electrophoresis and gel filtration. Under unfavourable conditions the natural blue complex, designated the α-form (approx. M r 246 000), dissociated to a purple dimer, the β-form (approx. 41 600). Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated the β-form to contain two polypeptides, of molecular weight 19 200 and 21 400. The amino-acid composition of the natural protein is described and compared with those of similar carotenoproteins from other crustaceans. The complex contains six carotenoid molecules per molecule of protein (α-form) and the carotenoid has been identified by thin-layer chromatography, light-absorption spectroscopy, HPLC and mass spectrometry as all- trans -astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione), the three optical isomeric forms, (3 R ,3′ R )-, (3 R ,3′ S ) and (3 S ,3′ S ), being present in the ratio 20:21:58. The binding of the carotenoid, astaxanthin (absorption maximum in acetone at 470 nm) to the apoprotein results in a marked red spectral shift of about 165 nm, giving rise to absorption maxima at 635 nm and 585 nm for the α- and β-forms, respectively, in 50 mM phosphate buffer (pH 7.5). The λ max of any sample is dependent upon the relative ratio of α and β forms present.

Structural characteristics of the carotenoids binding to the blue carotenoprotein fromProcambarus clarkii

A blue carotenoprotein from the crayfishProcambarus 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.

A red carotenoprotein from the carapace of the crayfish, Procambarus clarkii

Abstract 1. 1. A red carotenoprotein ( λ max = 482 nm) containing astaxanthin and astaxanthin esters was extracted and purified from the carapace of the crayfish Procambarus clarkii . 2. 2. The extraction was achieved in the presence of Triton X-100. 3. 3. The red carotenoprotein had a mol. wt of ca. 140,000 (gel filtration). Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated only a single polipeptide of 8600 daltons. 4. 4. The red carotenoprotein contained 0.166 mg of protein, 0.833 mg of lipids, 0.032 μg of N -acetylglucosamine and 0.178 μg of astaxanthin per one mg of carotenoprotein complex.

Chemical properties and denaturation of the blue carotenoprotein from Procambarus clarkii

Abstract 1. 1. The stability of the blue carotenoprotein from the carapace of the crayfish P. clarkii has been studied: its carotenoid astaxanthin appears much more stable when bound to the protein than free in solution; the carotenoprotein is stable within the 5.5–8.0 pH range and at temperatures below 30°C. The optimal conditions for its storage are high ionic strength and −18°C. 2. 2. The denaturation procesess unleashed by different agents: SDS, acetone, DMF and urea, are compared.

Characterization of a blue astaxanthin protein from the carapace of the crayfish Astacus leptodactylus

Abstract 1. 1. A blue carotenoprotein ( λ max = 634 nm) containing astaxanthin as prosthetic group, was extracted and purified from the carapace of the crayfish Astacus leptodactylus . 2. 2. The blue carotenoprotein contained (3 S ,3′ S )-astaxanthin, (3 R ,3′ S , meso)-astaxanthin and (3 R ,3′ R )-astaxanthin in relative ratio 38:41:21. 3. 3. The blue carotenoprotein had an approximate mol. wt of 440,000 (gel filtration) and 437,000 (gradient gel electrophoresis). 4. 4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated the presence of two polypeptides of 19,600 and 18,600 daltons, with different mobility in polyacrylamide gel electrophoresis in the presence of 6 M urea. 5. 5. At low ionic strength and in the presence of denaturing agents such as SDS, urea, extreme pH and heat, the blue complex showed a greater stability than most of the carotenoproteins studied to date.

A yellow carotenoprotein from the carapace of the crayfish Astacus leptodactylus

Abstract 1. 1. Yellow carotenoprotein (λmax = 385 nm) containing astaxanthin and zeaxanthin, in a 1:1 relative ratio, as prosthetic group, was extracted and purified from the exoskeleton of the crayfish Astacus leptodactylus. The complex contained 12 carotenoid molecules per molecule of protein. 2. 2. The carotenoprotein of approximate mol. wt 563,700 (gel filtration) showed four polypeptide bands of 195,000, 151,000, 126,000 and 95,000 Mr on SDS-polyacrylamide gel electrophoresis.

Relatedness between α-crustacyanin from the lobster Homarus americanus and the blue carotenoprotein from the crayfish Procambarus clarkii

1. 1. The high degree of structural homology between the blue carotenoprotein from the carapace of the crayfish Procambarus clarkii and α-crustacyanin, the blue carotenoprotein from the carapace of the lobster Homarus americanus has been proved, by a number of techniques currently used, to estimate the relatedness among proteins, such as: comparison of aminoacid contents, polyacrylamide gel electrophoresis in the presence of urea, and peptide mapping. 2. 2. The successful reconstitution of a hybrid α-carotenoprotein by mixing subunits—purified by chromatofocusing—from the two different carotenoid-protein complexes corroborated the relatedness between both carapace blue carotenoproteins.

Studies on the reconstitution ofP. clarkii blue carotenoprotein from its isolated subunits

A blue carotenoid-protein complex (λmax 635 nm) was extracted and purified from the carapace of the crayfishProcambarus 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 (λmax 545 nm) were obtained. It was found that both monomers are required in order to 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.

A blue carotenoprotein from Upogebia pusilla. Purification, characterization and properties

Abstract 1. 1. A blue carotenoprotein ( λ max = 622 nm ), containing astaxathin as the prosthetic group, was purified from the hypodermis and exoskeleton of Upogebia pusilla. 2. 2. The carotenoprotein of approximate molecular weight 363,500 (gel filtration) showed only one polypeptide band of 21,000 on SDS-polyacrylamide gel electrophoresis. 3. 3. The absorption spectra recorded at different pH and temperatures show that the carotenoprotein is stable within the 5.5–9.0 pH range and temperatures below 37°C.