Structural characterization of thermal and urea denaturation of the Pv2 apple snail perivitellin

FRASSA,V.; DREON, M.S.; CEOLIN,M.; HERAS, H.

Montevideo, Uruguay

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

The perivitellin fluid of the eggs of the snail Pomacea canaliculata is mostly composed of galactogen and several glycoproteins (perivitellins) being ovorubin and Pv2 the major ones with 60% and 10% of the protein content of the eggs, respectively. PV2 is an oligomer of 400 kDa, composed of two subunits of 67 and 31 kDa. The native Pv2 particle is a compact structure with a gyration radius of ~44Å, as revealed by Small Angle x-Ray Scattering (SAXS) experiments. Circular Dichroism (CD) analysis in the far UV region indicated an α+β secondary structure content for Pv2. Near UV CD experiments showed sharp spectroscopic features centered at 282 and 291 nm characteristic of buried tyrosines and tryptophan residues, respectively. Fluorescence Spectroscopy showed emission signals with a maximum below 340 nm confirming the presence of occluded tryptophan residues.Thermal stability was followed by tryptophan fluorescence emission, elipticity at 222 nm and evolution of gyration radius by SAXS. Clearly, Pv2 was stable up to 70ºC showing a normal thermal quenching of the fluorescence signal. Around 70ºC the fluorescence spectrum showed a small but noticeable red shift indicating exposure of the tryptophan residues to the solvent. Moreover, the CD melting curve also showed significant changes nearly 75ºC. The gyration radius increased from 44 to 61Å between 20 and 60ºC. All three techniques suggest the formation of high MW aggregates at 80ºC.The effect of increasing concentration of the chaotrope urea on Pv2 showed a strong red shift typical of solvent-exposed tryptophan residues in the fluorescence spectra. An unexpected rise of the intensity of the fluorescence signal would probably indicate the presence of internal quencher/s.Although Pv2 showed a higher sensitivity to thermal and urea denaturalization as compared with that of ovorubin, both perivitellins could have physiological meaning in protecting embryos exposed to temperatures up t o 50˚C attainable in eggs deposited on rocks above the waterline under direct sun radiation.