Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze-thaw treatment by compatible solute additives

In this study on M-4-lactate dehydrogenase (LDH) we were able to show that the addition of compatible solutes (glycine betaine, hydroxyectoine) shifts the enzyme's activity curve towards higher temperature. This increase in temperature stability is gained at the expense of a slightly reduced maximal activity and is also reflected in an increase in activation energy. In addition, tryptophan fluorescence spectroscopy has been used to monitor structural changes of the enzyme under conditions of freeze-thawing and urea treatment in the presence of a number of organic and inorganic solutes. As the data revealed that changes in fluorescence intensity are directly related to changes in enzyme activity, we were able to evolve a method for rapid assessment of enzyme stabilisation on the basis of fluorescence measurements. All organic solutes under investigation displayed remarkable stabilising properties, although the degree of stabilisation depended on both the type of solute and the stress factor chosen. It has to be noted that ammonium sulphate also performed very well as a stabiliser against heat and urea treatment, whereas the addition of inorganic salts during freeze-thawing apparently destabilises protein structure, at least under the test conditions employed. (C) 1999 Elsevier Science B.V. All rights reserved.