Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength
Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength
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DOI：
10.1016/j.bbapap.2013.06.001
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发表时间：
2013-10-01
影响因子：
3.2
通讯作者：
McManus, Jennifer J.
中科院分区：
文献类型：
--
作者：
Blumlein, Alice;McManus, Jennifer J.
DSC分析已经被用来定量溶菌酶热变性后去折叠的可逆性。由于蛋白质展开的温度T-m随溶液条件的不同而不同，因此考察了在低离子强度磷酸二糖缓冲液(5-1000 mm)中，在有无二糖存在的情况下，在pH(5-9)的范围内，对蛋白质的熔融温度和热变性后复性程度的影响。这项研究比较了连续加热循环中的展开热，以量化热变性后的可逆性。二糖、海藻糖和麦芽糖被用来评估双糖引起的T-m增加是否反映在热诱导变性的可逆性上。在发生不可逆热变性和可逆热变性的T-m值之间存在广泛的重叠。事实上，对于pH为6的最高和最低T-m，没有观察到复性，而对于中间值，观察到了复性，但具有相似的T-m值，具有不同比例的复性蛋白质。我们建立了一种方法来测量热变性后的可逆展开程度，从而间接地测量蛋白质丢失到不可逆聚集的程度，并表明提高熔体转变温度的溶液条件并不自动增加可逆性。这种类型的分析在评估生物制药和食品工业中蛋白质的稳定性方面可能被证明是有用的。(C)2013爱思唯尔B.V.保留所有权利。
DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T-m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5-1000 mM) over a range of pH (5-9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T-m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T-m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T-m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T-m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries. (C) 2013 Elsevier B.V. All rights reserved.