J Comp Physic B (1984) ~55:81-88 Journal of Comparative B,o,om,o,Bi~ and Environ- Physiology S mort,o, Physiology 9 Springer-Verlag 1984 Antifreeze polypeptides from the Newfoundland ocean pout, Macrozoarces americanus: presence of multiple and compositionally diverse components Choy L Hew 1, Don Slaughter 2 Shashikant B. Joshi 1, Garth L. Fletcher 3, and V.S. Ananthanarayanan 2 Department of Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G IX8, Canada 2 Department of Biochemistry and 3 Marine Sciences Research Laboratory, Memorial University of Newfoundland, St. Johns, Newfoundland AlE 3X9, Canada Accepted March 1, 1984 Summary. Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfound- land ocean pout. Except for their approximately identical sizes (6,000 Dalton), these components were shown to be separate entities by their behav- iour on polyacrylamide gel electrophoresis, ion ex- change chromatography, gel permeation and re- verse phase high performance liquid chromatogra- phy. They could also be divided into two cross- reactive, yet distinct, immunological groups. Ami- no acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not con- tain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 tool%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular anti- freeze. Introduction The blood plasma of a variety of teleost fishes from polar and subpolar oceans contains antifreeze gly- coproteins (AFGP) or antifreeze polypeptides (AFP). These macromolecules, which depress the freezing temperature of the body fluids, are essen- tial for the survival of these fish in ice laden seawa- ter (DeVries 1974; Feeney and Yeh 1978 ; Anan- thanarayanan and Hew 1978; Hew 1981; DeVries 1982). All of them lower the freezing temperature Abbreviations. AFGP antifreeze glycoprotein(s) ; AFP antifreeze polypeptide(s); HPLC high performance liquid chromatogra- phy; SDS sodium dodecyl sulfate; PAGE polyacrylamide gel electrophoresis in a noncolligative fashion and exhibit thermal hys- teresis (DeVries 1974; Feeney and Yeh 1978). AFGP have been isolated from the plasma of a number of fish species from the Antarctic (DeVries etal. 1970), Arctic (Raymond etal. 1975; Van Voorhies et al. 1978; Osuga and Feeney 1978) and North Atlantic oceans (Hew et al. 1981; Fletcher et al. 1982a). Apart from the number and size of the polymers as well as the presence of proline and arginine in some of the smallest components (Osuga and Feeney 1978; Morris et al. 1978; Hew et al. 1981 ; Fletcher et al. 1982a; O'Grady et al. 1982), the AFGP from all of the species studied to date have an identical repeating structure of alanylalanylthreonine, with a disaccharide moiety linked to the threonyl residue. In contrast, AFP isolated from the North Atlantic teleosts show con- siderable structural diversity. The AFP from the winter flounder, Pseudopleuronectes americanus, contains two major, and closely related, compo- nents of 4,000 Dalton. Both consist of nine differ- ent amino acids of which alanine accounts for 60% of the residues (Duman and DeVries 1976; Hew and Yip 1976; Davies et al. 1982). The AFP from the shorthorn sculpin, Myoxocephalus scorpius, is similar to that of the winter flounder with respect to the abundance of alanine in its amino acid com- position and its high c~-helical content (Ananthan- arayanan and Hew 1977). However, it contains 12 different amino acids and has at least three compo- nents (Hew et al. 1980). In contrast to the winter flounder and shorthorn sculpin, the AFP from the sea raven Hemitripterus arrtericanus contains all 20 of the normally occurring amino acids with only a modest amount of alanine (14%) and a high half- cystine content. In addition, sea raven AFP differs from the flounder and sculpin AFP in its secondary