Studies of the blue astaxanthin-proteins of velella velella (coelenterata: chondrophora)

Velella velella is a surface living oceanic chondrophore exhibiting the typical blue pigmentation of the neuston. In the present study the properties of the blue astaxanthinproteins of the mantle tissue are reported. The pigments have been separated into four main fractions by gel filtration (V620, V600, V570 and V545) with λmax 620 nm, 600 nm, 570 nm and 545 nm, respectively, and their molecular sizes were estimated. Pigment V620 is a polymeric form, possibly a tetramer, of pigment V600. Analysis of the gradient elution profile of V600 in gel filtration at low chloride ion concentration provides evidence that the pigment is an octamer of V570-size units. Pigment V570 separates in preparative acrylamide electrophoresis into six components. Gel filtration, SDS-electrophoresis and phase-partition experiments show that these are dimers of apoprotein (V545-size) units of molecular mass ca. 2.3 x 10 4 . Estimates of minimum molecular mass based on astaxanthin content reveal that V620 and V600 have two astaxanthin prosthetic groups per apoprotein subunit. The pigments are simple proteins with free sulphydryl groups and negligible tyrosine contents. Pigments V620 and V600 possess specific anion-binding sites which have an influence on the visible and circular dichroism spectra, and on the quaternary structure of the pigments. In the absence of halide ions the pigments dissociate reversibly forming pigments with λmax 583 nm (V600-size) and λmax 565 nm (V570-size). The aniondependence of the spectra of the native pigments and of the components, V583 and V565, separated in halide-free gel filtration, have been investigated. Equations are derived linking the spectral changes and anion-dependent association processes to the anion concentration. The order of effectiveness of anions for the changes (Br - > I - > Cl - > NO - 3 , CNS - ) does not follow the Hofmeister series. The changes take place at low anion concentrations (10 -5 -10 -1 M) with a negative temperature dependence and are partially abolished by blocking lysine residues; higher anion concentrations are required to bring about alterations in the spectra of the separated components V583 and V565. The halide-dependent quaternary structural changes of the pigments have been followed in gel filtration on Bio-Gel columns equilibrated with phosphate buffer containing low concentrations of KGl. In order to explain the high affinity of the pigments for anions and the order of anion effectiveness, it is proposed that the anion-binding sites consist of clusters of basic residues which impose stereospecific restrictions on the anion binding. These sites are progressively lost in the absence of halide ions and following gel filtration of halide-free solutions of the pigments. 36 Chloride-binding measurements, using a gel filtration equilibrium technique, indicate that V600 may have three such sites per apoprotein subunit. The temperature dependence of chloride binding and of the spectral alterations of the pigment show that occupation of the sites causes relatively minor changes in the polypeptide conformations of the native pigment; surface spreading of the pigment revealed only a small increase in the stability of the tertiary and quaternary structure of the pigment in the presence of chloride ions. Pigment V570 and the apoprotein do not possess halide-ion binding sites of high affinity. The astaxanthin prosthetic groups of pigments V620 and V600 are optically active with large molar ellipticity values in the visible region; pigment V570 shows a low molar ellipticity value at the visible absorption maximum. The origin of the optical activity of the carotenoid in the complexes is discussed. Exciton splitting, attributed to carotenoid-carotenoid interaction, is observed in the circular dichroism (c.d.) spectrum of V620 but only in the presence of halide ions. It is suggested that occupation of the anion-binding sites of the pigment affects the relative orientation or distance apart of the chromophores. A comparison of the c.d. spectrum of the pigments in the far ultraviolet did not reveal any major alteration in protein conformation resulting from anion binding. The c.d. spectrum of V600 in the far ultraviolet and the amino acid composition of the pigment are consistent with a high contribution of β-structure to the configuration of the protein. Investigations into the specificity of carotenoid attachment have shown that although astaxanthin combines most efficiently with the apoprotein, other carotenoids, including acetylenic and cyclopentene derivatives, are able to effect quaternary structural changes of the protein. The carotenoid effects dimerization of the apoprotein in the presence of a 4-keto group in one ring and the presence in either ring of an additional hydrophilic substituent; dimerization of the apoprotein is also obtained with the half-carotenoid, 15-15'-dehydro-8'-apo-β-carotene-8'-al-3,4-dione. Both 4- and 4'-keto groups and additional hydrophilic substituents in the carotenoid structure are prerequisites for the further polymerization of the protein. Garotenoproteins showing chloride-dependent spectral shifts are obtained only with carotenoids containing a keto substituent in both rings.