Lobster shell carotenoprotein organisation in situ studied by photoacoustic spectroscopy

Abstract

A SINGLE carotenoid pigment, astaxanthin, bound to specific proteins in a complex quaternary structure, provides shell coloration in the lobster Homarus americanus^1–8. The absorption of light by these carotenoproteins is uniform over a remarkable spectral range, as evidenced by the reflectance of a blue shell fragment which is low (5–6%) and essentially constant from 400 to 700 nm (ref. 9). This uniform absorption cannot be assigned to any combination of the carotenoprotein complexes which have been isolated. To assess the significance of these chemical species and to make specific inferences about carotenoprotein function in vivo, it is essential to measure the absorption spectrum in situ¹⁰. Several astaxanthin–protein complexes which absorb in the visible region have been isolated and spectroscopically characterised^1–9,11. These include α-and γ-crustacyanin with absorption maxima at 632 and 625 nm, respectively, and a second complex called the yellow protein^7,11 which absorbs maximally at 410 nm. An early in situ absorption measurement of a carapace sample taken immediately after ecdysis and before calcification has been reported by Cheesman et al.⁴. However, the absence of structure in the reflectance measurements⁹ from a mature, opaque carapace suggests that conventional spectroscopy can yield little information about absorption maxima in situ. By using a new spectroscopic technique, photoacoustic spectroscopy (PAS), we have obtained spectra of a native shell fragment and here report the existence of a near continuum of astaxanthin absorption energies which span the visible spectral region. Furthermore, these pigments are in an anisotropic distribution within the endocuticle, that is, those absorbing in the blue are near the surface whereas those absorbing in the red are in the interior.