Chromophore Content and Molar Absorptivity of Phytochrome in the Pr Form

Abstract

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 A⁶²⁰ _native/A^655-665 _degraded = 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 P_r were A_native/A_denatured = 3.4 and A_native/A_degraded = 3.7, this yielded ε⁶⁶⁵ _native = 109000 to 118000 and ε³⁸⁰ _native = 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.