Low Resolution Structure and pH stability of Ovorubin, a lipoglycocarotenoprotein from the eggs of Pomacea canaliculata

ITUARTE,S.; DREON, M.S.; CEOLIN,M.; HERAS, H.

Montevideo, Uruguay

Conferencia; International Conference of Biophysics of Proteins (ICBP); 2007

ICBP

Ovorubin is the major egg protein from the apple snail (Pomacea canaliculata) perivitellin fluid (PVF) that holds several biological functions such as an energetic and structural source for embryo development and protease inhibitor. Ovorubin also has the cofactor astaxanthin (3,3´-dihydroxy-β-β-carotene-4,4´-dione), a potent antioxidant in membranes, that protects the embryos from photoxidative damage and confers the eggs their characteristic pinkish-red colouration. It is a ca. 300 kDa lipo-glyco-carotenoprotein complex with a hydration density of 1.27g/ml, composed of three subunits of ca. 28, 32, and 35 kDa.The first low resolution model of ovorubin, as well as its stability at different pH was investigated using small angle X-ray scattering (SAXS), transmission electron microscopy (TEM), circular dichroism (CD), and fluorescence and absorption spectroscopy. The analysis of the scattering data led us to estimate that ovorubin presents a discoidal shape of 120 by 78 Å. These values were in good agreement with the size and shape of the particles observed in TEM images. The ovorubin structure was stable in a wide pH range ( 4.0-12.0), below pH 4.0 alterations in its gyration radius and partial lose of tertiary structure were detected. The absorption spectra of ovorubin at different pH values showed no changes in intensity and shape. Intrinsic fluorescence spectra of ovorubin at different pH values only showed tryptophan solvent exposure at pH 2.0.Finally, only subtle changes in the tertiary structure of ovorubin were detected by CD at pH values below 4.0. These results indicate that while the protonation of acidic residues affects the tertiary and quaternary structure of the protein, the ASX binding site remains unaltered. In conclusion our results suggest that low pH values are probably triggering the disassembling, but not the unfolding of the oligomer. The ovorubin stability in a wide pH range, adds to the previously reported unusually high thermal (>95˚C) and chemical stability. This is the first low resolution model of the global shape of an invertebrate egg carotenoprotein. The high stability against pH reinforces the idea that ovorubin is tailored to withstand a wide variety of conditions in order to play its key role in embryo protection during development.