Mi Yanli, Wood George, Thoma Laura
10777 Science Center Drive, La Jolla Laboratories, Pfizer Inc, San Diego, CA 92121, USA.
AAPS J. 2004 Sep 7;6(3):e22. doi: 10.1208/aapsj060322.
The purpose of this study was to explore the cryoprotection mechanisms of high molecular weight polyethylene glycols (PEGs) (eg, PEG 4000 and PEG 8000) on lactate dehydrogenase (LDH). Ultraviolet activity assays, circular dichroism (CD) spectroscopy, gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (14)C-PEG 4000 labeling and binding, and cryostage microscopic study were conducted. Different molecular weights and concentrations of PEGs in LDH formulations were treated by freeze-thawing. Higher molecular weights and concentrations of PEGs in LDH-PEG formulations obtained better activity and secondary structure recoveries of LDH after freeze-thawing. Insoluble aggregation of LDH was not observed in gel filtration studies. SDS-PAGE results suggested surface characteristic modifications of LDH by the larger molecular weight PEGs. The 14C-PEG 4000 labeling and binding study showed extensive nonspecific interactions between the PEG 4000 and LDH molecules in a concentration-dependent manner. The bound LDH-PEG 4000/free PEG 4000 ratio increased when LDH or PEG 4000 concentrations increased. Cryostage microscopic study showed that PEG 8000 delayed the ice crystallization and eutectic transition of LDH formulation. It appeared that multiple mechanisms were at work during PEGs' cryoprotection of LDH. It was unclear whether the delayed eutectic characteristics of PEGs contributed to LDH cryoprotection. The favorable interaction, rather than preferential exclusion, between LDH and PEGs (eg, 4000) cryoprotected LDH.
本研究的目的是探讨高分子量聚乙二醇（PEGs）（如PEG 4000和PEG 8000）对乳酸脱氢酶（LDH）的冷冻保护机制。进行了紫外活性测定、圆二色性（CD）光谱分析、凝胶过滤、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、（14）C-PEG 4000标记与结合以及低温显微镜研究。通过冻融处理LDH制剂中不同分子量和浓度的PEGs。LDH-PEG制剂中较高分子量和浓度的PEGs在冻融后能使LDH获得更好的活性和二级结构恢复。在凝胶过滤研究中未观察到LDH的不溶性聚集。SDS-PAGE结果表明较大分子量的PEGs对LDH的表面特性有修饰作用。14C-PEG 4000标记与结合研究表明PEG 4000与LDH分子之间存在广泛的非特异性相互作用，且呈浓度依赖性。当LDH或PEG 4000浓度增加时，结合的LDH-PEG 4000/游离PEG 4000的比例增加。低温显微镜研究表明PEG 8000延迟了LDH制剂的冰晶形成和共晶转变。似乎在PEGs对LDH的冷冻保护过程中有多种机制在起作用。尚不清楚PEGs的延迟共晶特性是否有助于LDH的冷冻保护。LDH与PEGs（如4000）之间的良好相互作用而非优先排斥对LDH起到了冷冻保护作用。
