Isolation and characterisation of crocodile and python ovotransferrins

Abstract

Transferrins play a major role in iron homeostasis and metabolism. In vertebrates, these proteins are synthesised in the liver and dispersed within the organism by the bloodstream. In oviparous vertebrates additional expression is observed in the oviduct and the synthesised protein is deposited in egg white as ovotransferrin. Most research on ovotransferrin has been performed on the chicken protein. There is a limited amount of information on other bird transferrins, and until our previous paper on red-eared turtle protein there was no data on the isolation, sequencing and biochemical properties of reptilian ovotransferrins. Recently our laboratory deposited ten new sequences of reptilian transferrins in the EMBL database. A comparative analysis of these sequences indicates a possibility of different mechanisms of iron release among crocodile and snake transferrin. In the present paper we follow with the purification and analysis of the basic biochemical properties of two crocodile (Crocodilus niloticus, C. rhombifer) and one snake (Python molurus bivittatus) ovotransferrins. The proteins were purified by anion exchange and hydrophobic chromatography, and their N-terminal amino-acid sequences, molecular mass and isoelectric points were determined. All three proteins are glycosylated and their N-glycan chromatographic profiles show the largest contribution of neutral oligosaccharides in crocodile and disialylated glycans in python ovotransferrin. The absorption spectra of iron-saturated transferrins were analysed. Iron release from these proteins is pH-dependent, showing a biphasic character in crocodile ovotransferrins and a monophasic type in the python protein. The reason for the different types of iron release is discussed.