CHYOI~IOIA)GY, 9, 469-472 (1!172) Freezing Behavior of Aqueous Solutions of Glycoproteins from the Blood of an Antarctic Fish1 J. G. DUMAN AND A. L. DEVRIES Physiological Research Laboratory, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92037 A series of glycoproteins has been isolated from the blood serum of the Antarctic, nototh- eniid fish, Trematomus borchgrevinki, which on a mob1 basis were found to depress the freezing point of water 200-500 times more effectively than an ideal solute (I). These glycoproteins enable T. borchgrevinki to survive in the ice- laden water of McMurdo Sound where water temperatures are never warmer than -l.S”C even in summer (5). The glycoproteins can be separated into eight distinct bands using acrylam- ide gel electrophoresis with a borate buffer (2). All eight groups consist of repeating units of 0 I tlisnccl~nridc and differ only in their molecular weight (rang- ing from 21,500 daltons for glycoprotein 3 to 2,600 daltons for glycoprotein 8) and the substi- tution of proline for some of the alanine residues in glycoproteins 7 and 8 (2, 4). Upon freezing, solutions of the glycoproteins have been shown to exhibit a thermal hysteresis, where by the freezing point differs from the melting point (1) when determined microscopically in a capillary tube according to t,he method of Ramsay and Brown (6). For example, the freezing point of a 10 mg/ml solution of glycoproteins 3, 4, and 5 is -0.75”C while the melting point is -0.05”C. From such dnt,a it is obvious that the freezing behavior of these solut,ions do not follow that of an ideal solution. In a recent paper (1) DeVries proposed that this unusual freezing behavior of sohnions of the glycoproteins could be explained by their being adsorbed onto the surface of ice crystals thereby preventing water molecules from joining t,he ice lattice, t,hus effectively re- moving the ice cryskds as nuclrntion sites. Such Received August 28, 1972. ’ Sur)ported by NSF Grant GV 27327. a mechanism is consistent with the thermal hys- teresis phenomenon since at the melting point of the solution internal disintegration of the ice lattice would occur as usual. In this paper data are presented which support this crystal surface adsorption theory. MATERIALS AND METHODS Freezing point-depressing glycoproteins were isolated from the blood serum of T. borchgrev- inki according to the method of DeVries et al. (2). Glycoproteins 1 through 5 were studied as their native mixture with most of the compo- nents being 3, 4 and 5. Glycoprot’eins 6, 7, and 8 were separated and studied individually. Inacti- vated freezing point-depressing glycoproteins were made by oxidation of the hydroxyl group of carbon 6 of the galactose residue to a car- boxy1 group according to the method of Shier et al. (8). The glycoproteins were also inactivated by treatment with 0.2 M sodium borate. Both the carboxyglycoproteins and the borate-complexed glycoproteins no longer possess the unusual freezing point-depressing activity of the native glycoproteins. Dextran T-40 and Dextran T-20 were ob- tained from Pharmacia Fine Chemicals and had molecular weights of 40,000 and 20,000 daltons, respectively. The concentrations of the glycopro- teins and dextrans before and after freezing were measured using phenol and concentrated sulfuric acid (3). One and one-half-milliliter aqueous so- lutions of these compounds were frozen in test tubes (6.0 mm i.d. x 100 mm long) in a Lauda WB-20 refrigerated alcohol bath. An external temperature control system was attached which made it possible to control the temperature to kO.Ol”C. The test solution was positioned in the bath so that 0.2 ml of the solution in the tube was above the bath level and therefore remained liquid. To avoid supercooling a small seed crys- tal was formed by spraying the bottom of the tube with Spray-Freeze before immersing the 469 Copyright 0 1973 by Academic Press, Inc. -411 rights of reproduction in :~ny form reserved.