Isolation of the Phytochrome Chromophore. The Cleavage Reaction with Hydrogen Bromide

Abstract

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.

Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium

“…The less stable Z-ethylidine isomer was reported to be generated from the E isomer by photoisomerization (24). The E-and Z-ethylidine phycocyanobilin isomers were the major and minor products, respectively, of phycocyanin chromophore cleavage by HBr in TFA (18). The methanolysis product also consists of two closely related bilins with identical mass (8).…”

“…1). Previous work has established that the major phycocyanin methanolysis product is of the E configuration (6,9,10,18,24). The less stable Z-ethylidine isomer was reported to be generated from the E isomer by photoisomerization (24).…”

“…Becuase they form the same ethylidine-free isomeric derivative mesobiliverdin, it is suggested that their difference probably resides in the configuration about the ethylidine group. The major product derived from methanolysis, which is the lateeluting component in reverse-phase HPLC, was previously determined to be the E-ethylidine isomer (6,9,10,18). The earlyeluting product derived from in vitro enzymic transformation of biliverdin would therefore be the Z-ethylidine isomer.…”

“…Further support for the identification of the early-and late-eluting products as the Z-and E-ethylidine phycocyanobilin isomers, respectively, is that the degree and direction of the peak absorbance wavelength differences are very similar to those reported for the corresponding isomers ofthe dimethyl esters offree phytochrome chromophore, phytochromobilin (24). The E isomer was reported to be thermodynamicallly more stable than the Z isomer (24) (8,18). Our methanolysis was carried out at a lower temperature (42°C), which might preserve more ofthe early-eluting phycocyanobilin.…”

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

“…In 1969, the bile pigment produced from the Pr chromophore by oxidative degradation was first analyzed, and components derived from the Band C-rings were identified (8). The degradation product from the A-ring was released and identified in a two-step reaction (9) after the isolation of the product from the D-ring (10). Finally, the chromophore of phytochrome was determined as phytochromobilin (P⌽B), which differs from phycocyanobilin (PCB) only by substitution of the D-ring ethyl group with a vinyl group (11).…”