Preparation and properties of crystalline biliverdin IXα. Simple methods for preparing isomerically homogeneous biliverdin and [14C]biliverdin by using 2,3-dichloro-5,6-dicyanobenzoquinone

Abstract

Amorphous isomerically pure biliverdin IX alpha is readily prepared in more than 70% yield by dehydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide under carefully controlled conditions. Crystalline biliverdin IX alpha and amorphous [14C]biliverdin can be obtained similarly in more than 40+ yield. The pure crystalline pigment was characterized by elemental analysis, methylation, chemical and enzymic reduction to bilirubin, i.r.- and u.v.-visible-absorption spectroscopy, n.m.r. spectroscopy and field-desorption mass spectrometry, and its solubility was determined. Under certain conditions, dehydrogenation, gave biliverdin contaminated with III alpha and XIII alpha isomers as a result of disproporationation of bilirubin. Formation of non-IX alpha isomers depends on the concentrations of the reagents and the order in which they are mixed, and occurs under neutral anaerobic conditions. Free-radical reactions probably are responsible, suggesting that the first step in the deydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide is formation of a bilirubin cation radical, rather than hydride ion abstraction.

Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin‐dependent heme oxygenase from Synechocystis sp. PCC 6803

“…Eluate fractions were transferred to methanol:HCl (49:1, v/v) for spectrophotometric analysis. Standard mesobiliverdin IXα was prepared by oxidation of mesobilirubin IXα (McDonagh and Palma, 1980). Affinity chromatography on Ni-chelating column A 1-ml HiTrap chelating column (Pharmacia Biotech Inc., Piscataway, NJ) was prepared by washing with 5 ml of water, then 5 ml of 50 mM NiSO 4 , then 5 ml of water, and finally with 5 ml of binding buffer (20 mM Tris-HCl, pH 7.9, 5 mM imidazole, 0.5 mM NaCl).…”

The Phytochrome-Deficient pcd1 Mutant of Pea Is Unable to Convert Heme to Biliverdin IX[alpha].

“…HPLC conditions were identical to those for BV IXa purification, except that the mobile phase was changed to ethanol-acetone-100 mM formic acid (25:65:10 [v/v]; mobile phase B) to increase the resolution between BV IXa and 3(Z)-P@B (Terry et al, 1995). All bilins were prepared as 1 mM stock solutions in DMSO, using the following molar absorption coefficients: 66,200 M cm-l at 377 nm for BV IXa (McDonagh and Palma, 1980), 47,900 M cm-1 at 374 nm for 3(E)-PCB (Cole et al, 1967), and 64,570 M cm-i at 386 nm and 38,020 M cm-l at 382 nm for 3(E)-P@B and B(Z)-P@B, respectively (Weller and Gossauer, 1980). Absorption spectrophotometry of bilin and heme samples was performed using a spectrophotometer (model U-3410; Hitachi, Tokyo, Japan).…”

Siderophore and haem iron use by Tritrichomonas foetus

“…7B). These features are characteristic of the reduction of biliverdin to bilirubin (McDonagh & Palma, 1980). The use of haemin iron by T. foetus will be reported fully elsewhere.…”