José Carlos G. Milicua

ISOLATION OF A YELLOW CAROTENOPROTEIN FROM CARROT

Abstract A yellow carotenoprotein was extracted from carrot roots with Triton X-100, and then purified to electrophoretic homogeneity. The M r of the carotenoid-protein complex was estimated to be 290 000 by gradient gel electrophoresis. The carotenoprotein contained seven carotenoids: phytoene (77.8%); phytofluene (6.8%) δ- carotene (7.8%) β-carotene (3.5%) α-carotene (2.3%) lutein (1.2%) and a carotenoid epoxide (0.5%).

Further studies on the blue carotenoprotein from Astacus leptodactylus

Abstract 1. 1. The isoelectric point from the blue carotenoprotein from the carapace of the crayfish Astacus leptodactylus was estimated by isoelectric focusing as 4.56 ± 0.05 (average value). 2. 2. The two subunits of the protein moiety were separated by chromatofocusing, showing isoelectric points of about 6.15 (A 1 ) and 5.3 (A 2 ). Both subunits were identified by 6 M urea-PAGE vs the ones obtained after urea treatment of a native carotenoprotein sample. 3. 3. The amino acid content of the whole carotenoprotein and each isolated subunits was determined.

Determination of carotenoid pigments in several tree leaves by reversed-phase high-performance liquid chromatography

Abstract A rapid and simple reversed-phase high-performance liquid chromatographic method has been used to analyse all carotenoid pigments from leaves of five different trees. The pigments were separated on an octadecylsilane radial compression column, using a mobile phase mixtures of acetonitrile, methanol-water and ethyl acetate in three isocratic steps. This method resolves all higher plant photosynthetic pigments, carotenoids and also chlorophylls in less than 15 min, while achieving a relatively good separation of trans-lutein—the major carotenoid—from its isomers zeaxanthin and cis-lutein.

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.

Impact of different dietary phospholipid levels on cholesterol and canthaxanthin lipoprotein-serum transport and muscle deposition in rainbow trout.

The study was designed to assess the effect of a progressive increase of dietary phospholipid (PL) levels in the transport of cholesterol and canthaxanthin by serum lipoproteins and their deposition in trout muscle. Three groups of 30 immature rainbow trouts, in triplicate, with a mean body weight of 195 g were fed three experimental diets containing 0, 4, and 8% extra PL contents for 6 weeks. The two major lipoprotein classes in rainbow trout were HDL and LDL. Both lipoproteins were the main transporters of serum canthaxanthin, whereas cholesterol was transported principally by LDL. Serum cholesterol contents remained constant, whereas serum canthaxanthin was increased when the PL amount augmented. In muscle, PL seemed not to have an effect on cholesterol and canthaxanthin deposition. Therefore, as an extra-PL contribution in the diet did not increase relative percentages of cholesterol and/or canthaxanthin in trout muscle, the results support the hypothesis that dietary extra-PL addition is not necessary to increase cholesterol and canthaxanthin and thus fish flesh pigmentation. However, a saturation effect of diet PL contents was found on muscle canthaxanthin deposition.

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.

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.

Human LDL structural diversity studied by IR spectroscopy.

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques. LDL has a single protein, apoB-100, that binds to a specific receptor. It is known that the failure associated with a deficient protein-receptor binding leads to plaque formation. ApoB-100 is a large single lipid-associated polypeptide difficulting the study of its structure. IR spectroscopy is a technique suitable to follow the different conformational changes produced in apoB-100 because it is not affected by the size of the protein or the turbidity of the sample. We have analyzed LDL spectra of different individuals and shown that, even if there are not big structural changes, a different pattern in the intensity of the band located around 1617 cm−1 related with strands embedded in the lipid monolayer, can be associated with a different conformational rearrangement that could affect to a protein interacting region with the receptor.

Low-density lipoprotein density determination by electric conductivity

The predominance of small dense low-density lipoprotein (LDL) particles is associated with an increased risk of coronary heart disease. A simple but precise method has been developed, based on electrical conductivity of an isopycnic gradient of KBr, to obtain density values of human LDL fraction. The results obtained can distinguish LDL density populations and their subfractions from different patients. These data were corroborated by Fourier transform infrared spectroscopy (FTIR) (structure) and light-scattering analyses (size).