The reversible denaturation and reduction with dithionite has been studied for the phycobiliproteins, C-phycocyanin (1) and allophycocyanin (2) from Spirulina platensis, and C-phycoerythrin (4) from Fremyella diplosiphon (both cyanobacteria). By treatment with sodium dithionite, the chromophores are selectively reduced at the central (C-l 0) methine bridge, producing pigments with bilirubinoid (X max =418 nm from 1 and 2), and vinylpyrrolic (X max = 300 nm from 4) chromophores. The extent of reduction is dependent on the state of the protein. The chromophores of denatured biliproteins are completely reduced at O.SmM dithionite. In the native pigments, dithionite concentrations up to 0.5M lead only to partial reduction thus forming products containing both reduced and oxidized chromophores (e.g. "phycocyanorubins" from 1 and 2). The reduction is non-statistical with respect to the different chiomophores present in 1 and 4, the chromophores absorbing at shorter wavelengths being preferentially reduced. Abbreviations:Pr, Pfr = Phytochrome in the red and far-red absorbing forms, respectively; 1/1620 umt or 1 ^563 un i* i s tnat amount of substance in 1 m/which has an absorbance of 1.00 in a cuvette of path-length = 1 cm at the indicated wavelength.

Bile Pigments, Protein Interaction, Conformation, DenaturationThe chromophore-protein interactions of C-phycocyanin (C-PC) from Spirulina platensis have been studied by following the partial and complete denaturation with UV-Vis spectroscopy. From comparison with published MO calculations, an elongated conformation of the chromophore is sug gested for native C-PC, a cyclic one for denatured C-PC. By means of partial denaturation, a step wise unfolding of the protein has been demonstrated. The presence of at least two sets of spectro scopically different diromophores is suggested from the partial denaturation and low temperature experiments.Phycocyanins and phycoerythrins are photosyn thetic light harvesting pigments of blue-green, red and cryptophytan algae, which contain bile-pigment chromophores covalently bound to p ro tein s2. The absorption of the various types of these pigments cover the spectral range between about 500 and 670 nm practically completely. In spite of this spec tral variety, however, the phycobiliproteins contain with only few exceptions just two chemically distinct chromophores: the blue phycocyanobilin ( la ) and the red phycoerythrobilin (2) *• 2> 3. Uninfluenced by the protein, the free bases of the two chromola : Rj = Protein; R2 = C^Hs lb : Ri = Protein; R2 = C2H3 2: R = Protein * The terms phycocyanobilin and phycoerythrobilin are used for compounds with the molecular structure of la and 2, which are characterized by a substituted ethylgroup at C-3. For a critical discussion, see ref.2.

The verdin-type Chromophore of denatured C-phycocyanin (1) from Spirulina platensis is reduced to the corresponding rubin (2a) by sodium borohydride. The structure assigned is in agreement with the uv-vis spectroscopic properties of the product and was deduced from model studies with free bile pigments. Analogous model studies using sodium dithionite demonstrated a two-fold reactivity for this reagent, leading to products which are both o f the rubin spectral type under the conditions tested. True rubins (10,22-dihydrobilindions) are formed in low yield only if an excess of reagent is used in methanol/water mixtures. It is accompanied by polar addition product(s) of the same spectral type, which are generally formed exclusively. In particular, no bilirubin was formed under the reaction conditions previously applied for the chemical modification of phycobiliproteins and phytochrome. From this finding and from the strikingly different properties of the borohydride and dithionite products, of phycocyanin upon renaturation, the dithionite product is suggested to be a rubinoid addition product (2b) rather than a hydrogenation product.In contrast to the dithionite addition product 2b of phycocyanin, the chromophore of the true phycorubin (2a) remains stable upon renaturation. The uv-vis spectral properties of the chromophore are not markedly different whether the apoprotein is in its native or denatured state. The different electrophoretic mobilities of native (renatured) phycocyanin compared to the renatured borohydride product suggest that these two have different protein conformations. The preparation of these phycorubins renders the extensive techniques of bilirubin chemistry applicable in the study of biliproteins.

The (4 Z, 10 Z, 15E)‐2,3‐dihydrobilindione 4, along with the fully unsaturated (E, Z, Z)‐analogue 8, has been prepared from the corresponding (Z, Z, Z)‐isomer by a variation of Falk's method (Falk et aL, 1980). The photochemical and acid‐catalyzed back‐reactions have been studied by UV‐vis and 1H‐NMR spectroscopy.

Abstract-(a) Native PEC from the cyanobacterium, Mastigocladus laminosus, and its isolated asubunit show photoreversibly photochromic reactions with difference-maxima around 502 and 570 nm in the spectral region of the a-84 phycoviolobilin chromophore. (b) Native PEC and its P-subunit show little if any reversible photochemistry in the 600-620 nm region, where the phycocyanobilin chromophores on the P-subunit absorb maximally. (c) Reversible photochemistry is retained in ureadenatured PEC at pH = 7.0 or pH 6 3. The difference maxima are shifted to 510 and 600 nm, and the amplitudes are decreased. An irreversible absorbance increase occurs around 670 nm (pH < 3).(d) The amplitude of the reversible photoreaction difference spectrum is maximum in the presence of 4-5 M urea or 1 M KSCN, conditions known to dissociate phycobiliprotein aggregates into monomers.At the same time, the phycocyanobilin chromophore(s) are bleached irreversibly. (e) The amplitude becomes very small in high aggregates, e.g. in phycobilisomes. ( f ) In a reciprocal manner, the phototransformation of native PEC leads to a reversible shift of its aggregation equilibrium between trimer and monomer. The latter is favored by orange, the former by green light. (8) It is concluded that the phycoviolobilin chromophore of PEC is responsible for reversible photochemistry in PEC, and that there is not only an influence of aggregation state on photochemistry, but also vice versa an effect of the status of the chromophore on aggregation state. This could constitute a primary signal in the putative function as sensory pigment, either directly, or indirectly via the release of other polypeptides, via photodynamic effects, or the like.