Biliverdin dimethyl ester (2) in freshly prepared ethanolic solution showed fluorescence maxima at 710 and 770 nm which were shifted to 725 and 806 nm on monoprotonation. % for 2 and 2H' was similar at room temperature (1.1 . and 2.7 .respectively), and it increased at 77 K to 5.0 .for 2 and to 2.6 . lo-' for 2H'. This acid effect was reversed on neutralization. The pK, values of 2 were 4.3 kO.1 at room temperature and ca. 5.3 at 77 K, and those of the fluorescent state were higher by ApK = 1.5 and 4.4, respectively. Certain differences between the absorption and fluorescence excitation spectra reflect either anomalous relaxation properties of excited *2 and *2Hf or non-identity of the emitting species with the predominant ground state absorbers, or both. The changes of QF with temperature and protonation of 2 are interpreted in terms of radiationless deactivation induced by twisting around the methine-ring bonds and by intramolecular proton exchange in 2. These conclusions are supported by fluorescence measurements of the pyrromethenes 4, 5 , and 8, the pyrromethenone 9, and bilirubin dimethyl ester (10). When a degassed neutral ethanolic solution of 2 was left to stand in the dark at room temperature, or when an acidic solution was neutralized, a new fluorescence maximum at 500 nm (excitation spectrum at 77 K: Lax = 470 nm) developed. This emission disappeared again on addition of acid. It is ascribed to 10-ethoxy-or 10-hydroxybilirubin dimethyl ester (12) whose formation by nucleophilic addition of solvent or trace water to 2 is reversed on protonation, affording 2H'.
Binding studies on bovine serum albumin (BSA) with bilirubin (BR) have been performed under physiological conditions using circular dichroism (CD) and electronic absorption spectroscopy (UV-VIS). Analysis of data obtained from solutions at varying BSA/BR ratios (R), overall concentrations and pH suggests the coexistence of two similarly strong complexes with 1 : 1 stoichiometry (K ass,M ca. 1 × 10 7 dm 3 mol Ϫ1 ). Further binding to BSA affords associates for which a 2 : 1 stoichiometry (K ass,B ca. 5 × 10 4 dm 3 mol Ϫ1 , pH = 7.4) is proposed. Results indicate that these aggregates comprise the main transport form of BR under physiological conditions. The role of the differently compounded BSA-BR complexes for controlling the concentration of unbound BR in the plasma is discussed. Preliminary experiments performed on human serum albumin (HSA) are in line with an analogous complexing behaviour. ‡ For an interpretation of CD spectra in terms of conformation of the BR backbone see comments on p. 2122.
The scope and limitation of the circular dichroism of racemates induced b y an optically active cosolute (ICD) are elaborated. Accordingly, the interpretation of ICD spectra depends upon the barrier t o interconversion of enantiomers, the degree of complexation, the availability of reference values and population and spectral differences of the species involved. The distinct nature of these factors may occasionally prevent a meaningful application of this technique in the field of molecular and chiral recognition. The relevance of the more general considerations is outlined and eventual implications for the ICD spectra of bilirubin-lXa (1) reported in the literature are provided by the bridged analogues 2, 3a and 3b. X
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