Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions
Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions
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DOI：
10.1002/bit.10612
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发表时间：
2003-06-20
影响因子：
3.8
通讯作者：
Foster, PR
中科院分区：
文献类型：
--
作者：
Cao, EH;Chen, YH;Foster, PR
在没有冷冻保护剂的情况下，研究了模型蛋白，LDH， ADH和过氧化氢酶的冷冻变性，使用微冷冻台在控制良好的冷冻和解冻速率下进行。大多数实验数据是通过使用酶浓度为0.025 g/l的稀释溶液的研究获得的。蛋白质活性恢复对冻融速率的依赖表现为相互独立的效应，即慢速冻结（冻结速率约为1℃/min）和快速解冻（解冻速率为10℃/min）产生更高的活性恢复，而快速冻结和慢速解冻对蛋白质的损害更严重。磷酸钾缓冲液使缓冲液的冻结浓度和pH变化最小，这一现象可归因于冻融过程中表面诱导的变性。在快速冷冻时（如冷冻速率为bbb20℃/min时），会形成较小的冰晶和较大的冰液界面表面积，这增加了蛋白质分子暴露于冰液界面的机会，从而增加了对蛋白质的损伤。在解冻过程中，再结晶过程会对蛋白质造成额外的损害。再结晶对被包裹的蛋白质施加额外的界面张力或剪切，从而对后者造成额外的损伤。当缓冲溶质参与冻结时，由于冻结过程中缓冲溶液pH的变化，冻结和解冻后蛋白质的活性恢复降低。然而，除了在极低的冻结速率下(
The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1degreesC/min) and fast thawing (at a thawing rate >10degreesC/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20degreesC/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (