Ross Henry Morris Hatley

The cold-induced denaturation of lactate dehydrogenase at sub-zero temperatures in the absence of perturbants

Lactate dehydrogenase; Enzyme denaturation, cold; pH; Temperature; Cryosolvent

Subzero-temperature preservation of reactive fluids in the undercooled state: II. The effect on the oxidation of ascorbic acid of freeze concentration and undercooling

The rate of oxidation of ascorbic acid has been measured in both frozen and undercooled solutions. A new interpretation is advanced for changes in the rate of ascorbic acid oxidation in freeze-concentrated solutions. The results obtained with undercooled solutions indicate a rate reduction in line with that predicted by the Arrhenius equation. It is also demonstrated that undercoohng provides a method for greatly extending the shelf life of reactive fluids.

Preservation of viable cells in the undercooled state

Previous studies into the mechanisms governing the freezing of cells in the absence of extracellular ice have been extended to develop a method for the preservation of viable cells in the undercooled state. Deep undercooling of cells is achieved by suspending fine droplets of the cells in oil to make an emulsion, thus minimizing initiation of extracellular ice nucleation. Attempts to preserve yeast cells, cultured sainfoin cells, and dissected shoot-tips (pea and potato) in this way are described. The main findings are that yeast cells can be preserved undercooled at -20 degrees C for at least 16 weeks with no detectable loss of viability, showing that -20 degrees C is a low enough temperature for inhibition of significant biochemical deterioration and that the emulsions are stable over long periods. In preliminary experiments, sainfoin cells survived 24 hr at -10 degrees C, and shoot-tips survived 48 hr at -10 degrees C. Sainfoin cells, conditioned by growth in medium supplemented with sorbitol, showed enhanced survival after exposure to low temperatures and a lower intracellular freezing point than control cells. Possible reasons for this are discussed.

Subzero-temperature preservation of reactive fluids in the undercooled state. I. The reduction of potassium ferricyanide by potassium cyanide.

Many reactions show enhanced rates at subzero temperatures due to freeze concentration. The reduction of potassium ferricyanide by potassium cyanide has been studied at subzero temperatures in both the undercooled and the frozen state. The pseudo-first-order rate constants calculated differ greatly from those in previous reports. A high degree of freeze concentration and supersaturation in frozen bulk solutions occurs. It has been clearly demonstrated that undercooled preservation provides a useful method for the long-term storage of reactive mixtures.

Stable enzymes by water removal

Abstract The most effective way of rendering proteins shelf stable is by drying. Freeze drying is currently the universal method of choice. The physical and chemical changes experienced by a product during freeze concentration and subsequent drying are analysed, and it is shown how effective processes can be devised. Alternative ways of water removal are briefly discussed.

Cold destabilisation of enzymes

The thermal stability profile of chymotrypsinogen has been investigated in the temperature range 230-340 K, with special emphasis on the phenomenon of cold instability. Differential scanning calorimetry was used to study the heat capacities of the native and denatured protein in undercooled solution and the results were combined with those obtained by spectrophotometry at ordinary temperatures. The partial heat capacities of both forms decrease with decreasing temperature, assuming negative values. In the experimentally accessible temperature range (above the spontaneous nucleation temperature of ice) the heat capacity difference delta C is found to be positive with a non-linear temperature dependence. delta C is predicted to change sign at some low temperature which cannot, however, be reached by experiment for chymotrypsinogen. In contrast to earlier studies, covering a much more limited temperature range and having to employ an additional destabilisation by means of pH and/or chaotropes, the present findings permit the construction of a more reliable thermodynamic stability profile and related properties. These differ in important details from those reported for other proteins, but based on measurements only in the neighbourhood of the heat-denaturation temperature. The thermodynamic characteristics are, however, in good agreement with earlier predictions and with recent low-temperature measurements on the tetrameric enzyme lactate dehydrogenase.