Purification and structural analysis of a type III antifreeze protein from the european eelpout Zoarces viviparus.

It has recently been reported that the eelpout Zoarces viviparus synthesizes a family of antifreeze proteins (AFP) similar in sequence to type III AFPs. A method has been set up to separate these antifreeze proteins from blood serum of this teleost species. A total of nine proteins with antifreeze activity have been isolated, several to a purity suited for NMR experiments. One of the proteins, Zvafp13, has been subject to partial structure determination by NMR. 1D- and 2D-H NMR analyses were carried out. In the 1D-experiments it was observed that the protein contained 28 slow-exchanging amides, suggesting a compact structure. The 2D-experiments were utilized to assign observed signals to specific amino acids. From TOCSY- and NOESY-experiments 35 out of a total of 66 amino acids were assigned. The amide exchange pattern, protein primary sequence, chemical shifts and NOE-cross-peaks between amides and -protons in the -sheets suggest that Zvafp13 structurally resembles the recombinant type III AFP rQAE m1.1.

Isolation and some characterisation of antifreeze protein from the European eelpout Zoarces viviparus.

The European eelpout Zoarces viviparus is a common inhabitant in the coastal areas of the eastern Atlantic Ocean and the Baltic region. At least 3 different antifreeze proteins were purified from Z. viviparus serum but more isoforms are most likely present. Two antifreeze proteins with molecular weights of approx. 6.5-7 kDa were characterised and found to share high similarity to the type III antifreeze proteins found in other members of the family Zoarcidae. The antifreeze activity of Z. viviparus antifreeze proteins is concentration dependent and showed a saturation effect when the protein concentration reached 30 mg.ml-1 (crude serum) and 8 mg.ml-1 (partly purified serum) respectively. Further the antifreeze activity was found to be dependent of the buffer osmolality resulting in increasing thermal hysteresis when buffer osmolality was raised from 0 to 1M.

Variations in antifreeze activity and serum inorganic ions in the eelpout Zoarces viviparus: antifreeze activity in the embryonic state.

The eelpout Zoarces viviparus is a common inhabitant in the shallow waters along the Danish coastline. Specimens were caught in the brackish (12-16 per thousand) Roskilde fjord where water temperatures range from >20 degrees C during summer to subzero in winter. The serum melting points found in Z. viviparus varied between -0.76 (September) to -0.94 degrees C (January). Eighty to 97% of the serum melting points could be attributed to sodium, chloride and potassium. Hysteresis freezing points showed seasonal variation varying from -0.83 (September) to -2.08 degrees C (February). Serum antifreeze activity showed a seasonal variation with high levels (>1.2 degrees C) in winter and low levels (<0.1 degrees C) during summer and autumn. Antifreeze proteins are responsible for this antifreeze activity. Antifreeze activity was also found in Z. viviparus during their embryological development in the female ovary. Embryo thermal hysteresis reached the maximum level (approx. 0.6 degrees C) during December and maintained this level until parturition in January. Antifreeze activity seems unaffected by diminishing ice crystal fractions at ice fractions below 0.1 whereas ice fractions above 0.1 caused a decline in antifreeze activity.