The cleavage of the bilin chromophore from C-phycocyanin with hydrogen bromide yields 3E-configurated phycocyanobilin (4) as the major and 3 Z-configurated phycocyanobilin (5) as the minor reaction product. The reaction of synthetic 3E-configurated phytochromobilin (2) with hydrogen bromide and methanol leads only to a methanol adduct at the C-18 side chain (7) whereas the same reaction with the 3Z-configurated phytochromobilin (3) leads to 7 and 2. The bilin chromophore was cleaved also from phytochrome after preparation of phytochromobilin peptides. The detection of 2 and 7 suggested that 3Z-and 3E-configurated phytochromobilin were the primary products of cleavage from phytochrome. A reaction scheme is given which can explain the results of the reaction with hydrogen bromide and methanol.

Chromopeptides were prepared from the Pfr form of phytochrome by pepsin digestion. After separation from colorless peptides and Pr peptides by column chromatography, spectral characteristics of this Pfr peptide were determined (λ1max = 610nm, λ2max = 380nm in acid methanol). Irradiation of Pfr peptide produces Pr peptide without liberation of a detectable compound. The Pfr peptide is more sensitive to oxidation and reduction than the Pr peptide. Oxidation with iodine and reduction with dithionite leads to partial chemoconversion of the Pfr peptide to the Pr peptide. The results favor the model of cis-trans isomerization for the Pr ⇌ Pfr transformation.

Quantitative absorption spectra in the visible and UV region were recorded for denaturated phytochrome (P r) and phytochromobilin peptides in comparison with native Pr. The method was tested with C-phycocyanin from Spirulina platensis. Based on known molar absorptivities for denaturated phycocyanin and suitable model compounds, and on the ratio A620native/A655-665degraded = 2.9, ^native was determined to be 102000 m-1 cm-1 for one phycocyanobilin chromophore in native phycocyanin. Likewise, Native was calculated to be 19000 M-1 cm-1. The corresponding ratios for Pr were Anative/Adenatured = 3.4 and Anative/Adegraded = 3.7, this yielded ε665native = 109000 to 118000 and ε380native = 36000 M-1 cm-1 for phytochromobilin in native P r. This value corresponds to one phytochromobilin per small phytochrome (60000 D) if data from the literature are corrected for the content of colorless proteins. The latter has been asessed from (i) the purity index (A280/A665) and (ii) the contribution of the phytochromobilin chromophore at 280 nm as derived from model compounds.

Abstract— The circular dichroism spectra of oat phytochrome were recorded. Qualitatively, the same spectra were found for large (360 kilodaltons) and small (60 kilodaltons) phytochrome. Quantitative CD data were reported for small Pr and Pfr (photoequilibrium mixture with 20% Pr) in tris buffer (native state) and in acid urea (denatured state). Further, the CD spectra of a phytochromobilinpeptide in acid solution with and without urea were recorded. Differences between the data in native and denatured state are discussed.

The circular dichroism spectra of oat phytochrome were recorded. Qualitatively, the same spectra were found for large (360 kilodaltons) and small (60 kilodaltons) phytochrome. Quantitative CD data were reported for small P, and PI, (photoequilibrium mixture with 20'1~ P,) in tris buffer (native state) and in acid urea (denatured state). Further, the CD spectra of a phytochromobilinpeptide in acid solution with and without urea were recorded. Differences between the data in native and denatured state are discussed.