Global shape and pH stability of Ovorubin, an oligomeric protein from the eggs of Pomacea canaliculata

DREÓN, M.S.; ITUARTE, S.; CEOLÍN, M.; HERAS, H.H.

FEBS Journal

Año: 2008 vol. 275 p. 4522 - 4530

Ovorubin, a 300 KDa thermostable oligomer, is the major egg protein from the perivitellin fluid that surrounds the embryos of the apple snail Pomacea canaliculata. It plays essential functions for embryo development including transport and protection of carotenoids, protease inhibition, photoprotection, storage and nourishing. Here we report ovorubin dimensions and global shape and test the role of electrostatic interactions on conformational stability by analyzing the effects of pH, using small angle X-ray scattering (SAXS), transmission electron microscopy, circular dichroism, fluorescence and absorption spectroscopy. Analysis of SAXS data showed that ovorubin is an anisometric particle with a major axis of 130Å and a minor one varying between 63 and 76Å. The particle shape was not significantly affected by the absence of the cofactor astaxanthin. The 3D model presented here is the first for an invertebrate egg carotenoprotein. The quaternary structure was stable under a wide pH range (4.5-12.0). At pH values between 2.0 and 4.0 a reduction in the gyration radius and a loss of tertiary structure were observed, though astaxanthin binding was not affected and only minor alterations in secondary structure were observed. In vitro pepsin digestion indicates that OR is resistant to this protease action. The high stability over a considerable pH range and against pepsin, together with the capacity to bear up temperatures >95°C, 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.