1 78 Simon Freed 



In analogy with the phenomena illustrated we would expect that a knowledge 

 of biochemical and even biological processes of considerable value may be 

 gained by investigations at low temperature. Support for these expectations 

 comes mainly from recent investigations directed toward the preservation of 

 cells, tissues, and entire organisms. Even more cogent for our purposes are 

 the instances of partial preservation at low temperatures which becomes more 

 effective at still lower temperatures. Unless explicit references are given, the 

 following examples are drawn from the excellent review by Audrey U. Smith (3). 

 For example, H. F. Smart found that twenty-one species of bacteria, yeasts, 

 and molds continued to multiply in frozen media at 264. 1°K. Sizer and Joseph- 

 son found that lipase was active at 248. 5°K, tryptic digestion proceeded at 

 258°K, and that invertase continued to hydrolyze sucrose at 255°K. At 203°K, 

 however, they could detect no hydrolysis during several weeks. In the preser- 

 vation of red blood cells, about ten per cent deterioration occurs per year at 

 dry ice temperature, 193°K, but scarcely any loss is incurred when they are 

 kept at the temperature of liquid air, 80°K. Ovarian tissue failed to survive 

 nine days at 193°K but survived more than a year at 80°K under otherwise 

 similar conditions (4). Revival of rats after cooling to 273. 5°K was reported 

 by Andjus (5, 6). 



Irreversible reactions are then clearly progressing at low temperatures, 

 in red blood cells and ovarian tissue at 193''K and at somewhat higher tempera- 

 tures in the enzymatic reactions. If the simple reactions such as those of isoprene 

 and iodine, chlorophyll and propylamine serve as models, the irreversible 

 reactions are preceded in their first and intermediate stages by reversible reactions 

 at still lower temperatures.* 



Becquerel found that rotifers, spores of bacteria, non-sporing bacteria, 

 algae lichens, mosses, and seeds of higher plants, after having been dried in 

 a vacuum of 10^^ mm Hg over barium oxide, could be successfully kept at the 

 temperature of liquid helium (4°K). Parkes showed that human spermatozoa 

 survived exposure and storage at 80°K. Ovarian, testicular, pituitary, and 

 adrenal tissue have given functional grafts after storage at 80°K, especially 

 if glycerine was added. Luyet established that vinegar eels, spermatozoa 

 muscle fibres of frogs, and hearts of embryonic chicks could be revived after 

 sudden cooling to the temperature of liquid air (80°K). It is then not surprising 

 that enzymes have been cooled to such temperatures without loss of subsequent 

 potency. It would seem then that a number of biochemical and biological 

 processes are available for study at low temperatures. 



I shall consider both homogeneous and heterogeneous solutions. The 

 first implies that solvents must maintain all the reactants in solutions fluid 

 at low temperatures. It would seem well worthwhile to employ conventional 

 solutions at as low temperatures as possible, and aqueous systems near zero 

 degrees or under supercooled conditions. It has been shown (8) that proteins 



* Lovelock (7) ascribes the deterioration of red cells to a physical mechanism rather than to a 

 chemical process, namely, that the dissolution of lipoprotein and other components of the cell 

 membrane proceeds more rapidly than the biochemical processes can repair them at the low 

 temperature. Since the lipoprotein etc. is presumably bound as an integral part of molecules 

 composing the membrane material, the physical process may also be initiated by reversible 

 chemical transformations. 



