Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container.
Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container.
复制标题
DOI：
10.1016/j.btre.2015.03.004
复制
发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Nidetzky B
中科院分区：
文献类型：
--
作者：
Roessl U;Leitgeb S;Nidetzky B
在实验室冷冻容器中研究牛血清白蛋白的冷冻浓缩。通过共聚焦激光扫描显微镜观察蛋白质显微分离。冷冻方案影响蛋白质冷冻浓缩。冷冻浓缩造成的空间不均匀性。凝固参数对显微偏析有影响。为了研究不同冷冻条件对冷冻蛋白质溶液中空间异质性发展的影响，在温控的200 ml冷冻容器中研究了牛血清白蛋白（BSA）的宏观冷冻浓缩和微观分离。冷冻至-40 °C促进了从12个不同冷冻位置采集的冷冻样本中蛋白质浓度梯度（69-114 μg ml-1）的形成，其中4小时或更长时间内的缓慢冷冻促进了强烈空间异质性的演变，并导致局部浓度增加相对于初始蛋白质浓度（100 μg ml-1）的1.15倍。可视化蛋白质的微分离过程中相分离，BSA与异硫氰酸荧光素和共聚焦激光扫描荧光显微镜被用来定位和大小的冷冻浓缩的蛋白质区域。缓慢冷冻导致明显更少和更大的蛋白质结构域的冷冻散装比快速冷冻。因此，在低冷却速率下，冷冻过程中蛋白质的表面应力将最小化;然而，在这些条件下，微观冷冻浓度将最高，可能有利于蛋白质聚集。
Freeze concentration of bovine serum albumin studied in a laboratory freeze container. Protein micro-segregation visualized by confocal laser scanning microscopy. Freezing protocol affected protein freeze concentration. Spatial heterogeneities created by freeze concentration. Micro-segregation was affected by freezing parameters. To examine effects of varied freezing conditions on the development of spatial heterogeneity in the frozen protein solution, macroscopic freeze concentration and micro-segregation of bovine serum albumin (BSA) were investigated in a temperature-controlled 200-ml freeze container. Freezing to −40 °C promoted formation of protein concentration gradients (69–114 μg ml−1) in frozen samples taken from 12 different freezer positions, whereby slow freezing in 4 h or longer facilitated the evolution of strong spatial heterogeneities and caused local concentration increases by 1.15-fold relative to the initial protein concentration (100 μg ml−1). To visualize protein micro-segregation during phase separation, BSA was conjugated with fluorescein isothiocyanate and confocal laser scanning fluorescence microscopy was used to localize and size the freeze-concentrated protein regions. Slow freezing resulted in distinctly fewer and larger protein domains in the frozen bulk than fast freezing. Surface stress on the protein during freezing would therefore be minimized at low cooling rates; microscopic freeze concentration would however be highest under these conditions, potentially favoring protein aggregation.