Mechanisms of spectral shifts in lobster carotenoproteins The resonance raman spectra of ovoverdin and the crustacyanins

Abstract Resonance Raman data have been used to elucidate the mechanisms of the absorption spectral shifts occurring for astaxanthin upon binding to the carotenoproteins, ovoverdin and α-, β- and γ-crustacyanins, from the lobster Homarus americanus . Although distinguishable on the basis of small differences in their resonance Raman spectra the binding sites of the crustacyanins, giving rise to λ max at 605 ± 25 nm, are essentially the same. The large red shift in λ max for the crustacyanins compared to free astaxanthin ( λ max 480 nm), is accounted for by a charge-polarisation mechanism in which charged groups and possibly hydrogen bonds in the binding site set up π electron polarisation in the ligand. Several alternate mechanisms can be eliminated. Ovoverdin is found to consist of three polypeptide chains of molecular weight 105 000, 95 000 and 78 000 which are not linked by disulfide bridges. The visible absorption peaks of ovoverdin at 460 and 640 nm are shown to arise from two astaxanthin molecules each bound at a different site. The spectral characteristics of the 460 nm site suggest a rigid hydrophobic environment for astaxanthin, in which no charge-ligand interactions occur. The mechanism of the spectral shift in the 640 nm site is the same as in the crustacyanins, i.e. a charge-polarisation effect. Resonance Raman spectra of ovoverdin and the crustacyanins could be obtained in situ; they were identical to the spectra of the purified proteins showing that the carotenoid sites were unperturbed by protein isolation.