KINETICS OF UNFOLDING OF PROTEINS ON HYDROPHOBIC SURFACES IN REVERSED-PHASE LIQUID-CHROMATOGRAPHY
KINETICS OF UNFOLDING OF PROTEINS ON HYDROPHOBIC SURFACES IN REVERSED-PHASE LIQUID-CHROMATOGRAPHY
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DOI：
10.1016/s0021-9673(01)91662-0
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
KARGER, BL
中科院分区：
文献类型：
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作者：
BENEDEK, K;DONG, S;KARGER, BL
本文在前人研究的基础上，研究了木瓜蛋白酶、大豆胰蛋白酶抑制剂和溶菌酶在正丁基键合硅胶表面上的变性动力学。在所有情况下，均观察到天然峰和变性峰彼此分离。变性或解折叠的速率常数通过测量天然蛋白质的峰面积来确定，所述天然蛋白质的峰面积是物种在键合相表面上花费的孵育时间的函数。结果表明，一个缓慢的变性步骤发生的半衰期约。15分钟。此外，变性的研究作为1-丙醇在初始移动的相的量的函数建议一个额外的展开步骤时，蛋白质接触的键合相表面。这后一步的程度随着1-丙醇浓度的增加而降低，进一步表明1-丙醇在键合固定相上的吸附可能在此行为中起作用。其他研究使用α-胰凝乳蛋白酶原进行，其中在梯度开始后进样。观察到变性程度是所用有机改性剂的函数。本文的结果提供了深入了解变性过程中的RPLC和建议的方法，以尽量减少这种行为。
As a continuation of previous studies, we present in this paper measurements on the kinetics of denaturation of papain, soybean trypsin inhibitor and lysozyme onn-butyl-bonded silica gel surfaces used in reversed-phase liquid chromatography (RPLC). In all cases, native and denatured peaks widely separated from one another are observed. The rate constants for denaturation or unfolding are determined by the measurement of the peak area of the native protein as a function of the incubation time that the species spends on the bonded-phase surface. The results reveal that a slow denaturation step occurs with a half-life ofca.15 min. In addition, studies of denaturation as a function of the amount of 1-propanol in the initial mobile phase suggest an additional unfolding step when the protein comes in contact with the bonded-phase surface. The extent of this latter step decreases as the concentration of 1-propanol increases, further suggesting that 1-propanol sorption on the bonded stationary phase may play a role in this behavior. Other studies are conducted with α-chymotrypsinogen, in which injection is made after the start of the gradient. The extent of denaturation is observed to be a function of the organic modifier employed. The results of this paper provide insight into the denaturation process in RPLC and suggest approaches to minimize this behavior.