Allison S D, Chang B, Randolph T W, Carpenter J F
University of Colorado Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, Denver, Colorado, 80262, USA.
Arch Biochem Biophys. 1999 May 15;365(2):289-98. doi: 10.1006/abbi.1999.1175.
The nature of the interaction responsible for the inhibition of protein unfolding and subsequent damage by sugars during dehydration is unclear. The relationship between sample moisture content measured by coulometric Karl Fischer titration and the apparent moisture content predicted by the area of the protein side chain carboxylate band at approximately 1580 cm-1 in infrared spectra of dried protein-sugar samples was examined. For samples in which a high level of native protein structure was retained in the dried solid, the apparent moisture content predicted by the carboxylate band area was greater than the actual moisture content, indicating that protection results from direct sugar-protein hydrogen bonding and not entrapment of water at the protein surface. Further, we show that the degree of structural protection conferred by sucrose and trehalose apparent in second derivative, amide I infrared spectra, correlates with the extent of hydrogen bonding between sugar and protein. The failure of dextran to inhibit dehydration-induced lysozyme unfolding is shown to result from the inability of the polymer to hydrogen bond adequately to the protein. Therefore, formation of an amorphous phase alone is not sufficient to maintain protein structure during dehydration. Glucose hydrogen bonds to a high degree with dried lysozyme, but is incapable of inhibiting lyophilization-induced protein unfolding in the absence of an effective cryoprotectant. However, the addition of polyethylene glycol, which is known to protect proteins during freezing, but not drying, to glucose protected lysozyme structure during lyophilization. Together, these results show that hydrogen bonding between carbohydrate and protein is necessary to prevent dehydration-induced protein damage. However, hydrogen bonding alone is not sufficient to protect proteins during lyophilization in the absence of adequate freezing protection.
脱水过程中糖类抑制蛋白质解折叠及后续损伤的相互作用本质尚不清楚。研究了通过库仑卡尔费休滴定法测得的样品水分含量与干燥蛋白质 - 糖样品红外光谱中约1580 cm-1处蛋白质侧链羧酸盐带面积预测的表观水分含量之间的关系。对于在干燥固体中保留高水平天然蛋白质结构的样品，由羧酸盐带面积预测的表观水分含量大于实际水分含量，这表明保护作用源于糖 - 蛋白质直接氢键作用，而非蛋白质表面水的截留。此外，我们表明，在二阶导数酰胺I红外光谱中蔗糖和海藻糖赋予的结构保护程度与糖和蛋白质之间的氢键作用程度相关。葡聚糖未能抑制脱水诱导的溶菌酶解折叠，这表明该聚合物无法与蛋白质充分形成氢键。因此，仅形成无定形相不足以在脱水过程中维持蛋白质结构。葡萄糖与干燥的溶菌酶高度形成氢键，但在没有有效冷冻保护剂的情况下无法抑制冻干诱导的蛋白质解折叠。然而，添加已知在冷冻过程中保护蛋白质但在干燥过程中不行的聚乙二醇，可在冻干过程中保护溶菌酶结构。总之，这些结果表明碳水化合物与蛋白质之间的氢键作用对于防止脱水诱导的蛋白质损伤是必要的。然而，在没有充分冷冻保护的情况下，仅靠氢键不足以在冻干过程中保护蛋白质。
