The Effect of Stabilizers and Denaturants on the Cold Denaturation Temperatures of Proteins and Implications for Freeze-Drying
The Effect of Stabilizers and Denaturants on the Cold Denaturation Temperatures of Proteins and Implications for Freeze-Drying
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DOI：
10.1007/s11095-005-6035-4
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发表时间：
2005-07
影响因子：
3.7
通讯作者：
X. Tang;M. Pikal
中科院分区：
文献类型：
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作者：
X. Tang;M. Pikal
目的研究稳定剂和变性剂对冷冻干燥系统中选定蛋白质的热变性和冷变性温度的影响。方法选择β-乳球蛋白和磷酸甘油酸激酶（PGK）作为模型蛋白。用常规差示扫描量热法和调制差示扫描量热法测定了蛋白质的热变性和冷变性温度，并用色氨酸发射光谱在选定的体系中进行了验证。结果β-乳球蛋白的冷变性是可逆的，而热变性只有在高扫描速率（10°C/min）下才可逆。β-乳球蛋白的冷变性温度随蛋白浓度的增加而降低（自稳定）。冷变性温度随着pH值的增加而升高（从pH 2到pH 7），每单位pH变化约增加4.6℃。所研究的所有稳定剂（即蔗糖、海藻糖和甘油）都提高了所研究蛋白质的热变性温度，降低了冷变性温度。蔗糖降低PGK冷变性温度[每摩尔浓度增加40°C(40°C/M)]的作用与β-乳球蛋白（36°C/M）的作用相同。稳定剂对冷变性温度的影响远大于对热变性温度的影响。当蔗糖浓度为0 ~ 2.7 M时，β-乳球蛋白热变性温度仅升高约5℃，而当蔗糖浓度低至0.9 M时，冷变性温度的降低幅度超过35℃。与16°C/M （β-乳球蛋白上的盐酸胍）的文献数据相比，9°C/M （β-乳球蛋白上的尿素oncd2）和65°C/M （PGK上的盐酸胍）。β-乳球蛋白和PGK的冷变性温度分别为- 14°C和- 26°C。结论蛋白冷变性温度与pH、蛋白浓度和添加剂有关。稳定剂，如糖和/或多元醇，可以稳定蛋白质的热变性和冷变性，而变性剂会破坏蛋白质的冷变性。蛋白质冷变性的稳定作用远大于热变性，因此在蛋白质冷冻干燥中具有重要意义。
PurposeThe aim of the study is to investigate the effects of stabilizers and denaturants on the thermal and cold denaturation temperatures of selected proteins in systems of interest to freeze-drying.Methodsβ-Lactoglobulin and phosphoglycerate kinase (PGK) were chosen as model proteins. Protein thermal and cold denaturation temperatures were determined by both conventional and modulated differential scanning calorimetry and verified by tryptophan emission spectroscopy in selected systems.ResultsThe cold denaturation of β-lactoglobulin was reversible, whereas the thermal denaturation was only reversible at high scanning rate (10°C/min). The cold denaturation temperatures of β-lactoglobulin decreased with an increase in protein concentration (self-stabilization). The cold denaturation temperature increased with increases in pH (from pH 2 to 7) with about 4.6°C increase per unit pH change. All stabilizers studied (i.e., sucrose, trehalose and glycerol) increased the thermal denaturation temperature of the proteins studied and decreased the cold denaturation temperature. The effect of sucrose in decreasing the PGK cold denaturation temperature [40°C per molar concentration increase (40°C/M)] was of the same magnitude as for β-lactoglobulin (36°C/M). The effect of stabilizers on cold denaturation temperatures is much greater than the effect on thermal denaturation temperatures. With sucrose, the β-lactoglobulin thermal denaturation temperature increases only about 5°C from 0 to 2.7 M, whereas the decrease in cold denaturation temperature was more than 35°C even at sucrose concentrationsas low as 0.9 M. Denaturants (urea and guanidine hydrochloride) increased the cold denaturationtemperatures of proteins and thereby destabilized protein; the magnitudes were 9°C/M (urea onTcdof β-lactoglobulin) and 65°C/M (guanidine hydrochloride on PGK) compared with literature data of 16°C/M (guanidine hydrochloride on β-lactoglobulin). The cold denaturation temperatures of β-lactoglobulinand PGK extrapolated to zero concentration of denaturants were −14 and −26°C, respectively.ConclusionsThe protein cold denaturation temperature was pH-, protein concentration-, and additive-dependent. Stabilizers, such as sugars and/or polyols, can stabilize both protein thermal and cold denaturation, whereas the denaturants destabilize protein cold denaturation. The stabilization effect on protein cold denaturation is much larger than on thermal denaturation, a result of great importance in protein freeze-drying.