Global shape and pH stability of ovorubin, an oligomeric protein from the eggs of Pomacea canaliculata.
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 roles in embryo development, including transport and protection of carotenoids, protease inhibition, photoprotection, storage, and nourishment. Here, we report ovorubin dimensions and global shape, and test the role of electrostatic interactions in conformational stability by analyzing the effects of pH, using small-angle X-ray scattering (SAXS), transmission electron microscopy, CD, and fluorescence and absorption spectroscopy. Analysis of SAXS data shows that ovorubin is an anisometric particle with a major axis of 130 A and a minor one varying between 63 and 76 A. 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 is stable over a wide pH range (4.5-12.0). At a pH between 2.0 and 4.0, a reduction in the gyration radius and a loss of tertiary structure are observed, although astaxanthin binding is not affected and only minor alterations in secondary structure are observed. In vitro pepsin digestion indicates that ovorubin is resistant to this protease action. The high stability over a considerable pH range and against pepsin, together with the capacity to bear temperatures > 95 degrees 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.
Dreon
Marcos S
MS
Instituto de Investigaciones Bioquímicas de La Plata INIBIOLP, CONICET-UNLP, Argentina.
Ituarte
Santiago
S
Ceolín
Marcelo
M
Heras
Horacio
H
D000818:Animals; D004527:Egg Proteins / Q000737:chemistry* / Q000648:ultrastructure; D049851:Gastropoda / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010063:Ovum / Q000737:chemistry; D011487:Protein Conformation; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D004527:Egg Proteins; C002008:ovorubin