Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze-thaw treatment by compatible solute additives
Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze-thaw treatment by compatible solute additives
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DOI：
10.1016/s1381-1177(99)00043-0
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发表时间：
1999-09-15
影响因子：
--
通讯作者：
Galinski, EA
中科院分区：
文献类型：
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作者：
Göller, K;Galinski, EA
在对M-4-乳酸脱氢酶(LDH)的研究中，我们能够证明相容溶质(甘氨酸甜菜碱、羟基胞苷)的加入使酶的活性曲线向更高的温度移动。这种温度稳定性的提高是以略微降低最大活度为代价的，也反映在活化能的增加上。此外，还利用色氨酸荧光光谱监测了冻融条件下酶的结构变化，以及在多种有机和无机溶质存在下尿素处理的情况。由于数据显示荧光强度的变化与酶活性的变化直接相关，我们能够发展出一种基于荧光测量的快速评估酶稳定性的方法。所有研究中的有机溶质都表现出显著的稳定性能，尽管稳定的程度取决于溶质的类型和所选择的应力因子。必须指出的是，硫酸铵作为抗热和尿素处理的稳定剂也表现得很好，而在冻融过程中添加无机盐显然会破坏蛋白质结构的稳定，至少在所用的测试条件下是这样。(C)1999 Elsevier Science B.V.保留所有权利。
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.