Carbodiimide Induced Cross-Linking, Ligand Addition, and Degradation in Gelatin
Carbodiimide Induced Cross-Linking, Ligand Addition, and Degradation in Gelatin
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DOI：
10.1021/mp5006118
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发表时间：
2015-03-01
影响因子：
4.9
通讯作者：
Ofner, Clyde M., III
中科院分区：
文献类型：
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作者：
Cammarata, Christopher R.;Hughes, Mitchell E.;Ofner, Clyde M., III
水溶性碳二亚胺(1-ethyl-3-(3-(dimethylaminopropyl)-carbodiimide)在蛋白质化学中有着广泛的应用。我们使用EDC诱导的明胶交联作为酰胺键形成的模型，以解决与缓冲液、明胶浓度和pH等共同变量的反应模糊性。用SEC高分子量峰面积的百分比变化来跟踪反应。观察到四种常用缓冲液的反应速度和程度的差异，以及常用浓度和pH值的程度的差异。我们还研究了水溶液中EDC诱导的酰胺键形成的酸酐机制，自1995年提出以来，该机制几乎没有引起人们的注意。明胶羧基在联氨加成过程中起到协同作用，证实了羧基之间的酸酐生成。利用SEC低分子量峰面积的百分比变化研究了EDC对明胶的降解作用。在中性到碱性的pH下，过量的EDC发生降解，当反应的氨基不可用时，降解作用显著增强。提出了EDC诱导明胶降解的机理，命名为扩展的Khorana机理。这种EDC副反应有可能在类似条件下发生在多肽和蛋白质中。
The water-soluble carbodiimide, 1-ethyl-3-(3-(dimethylaminopropyl)-carbodiimide (EDC) is widely used in protein chemistry. We used EDC-induced gelatin cross-linking as a model for amide bond formation to resolve reaction ambiguities with common variables of buffers, gelatin concentration, and pH. Percentage changes in SEC high molecular weight peak areas were used to follow the reactions. Differences in reaction rate and extent were observed with four commonly used buffers, while differences in extent were observed for commonly used concentrations and pH. We also investigated an anhydride mechanism for aqueous EDC-induced amide bond formation that has received little attention since its proposal in 1995. Gelatin carboxyl groups had a synergistic role during the addition of hydrazine to corroborate the anhydride formation between carboxyl groups. EDC-induced degradation of gelatin was investigated using percentage changes in SEC low molecular weight peak areas. The degradation occurred in excess EDC at neutral to alkaline pH and was enhanced substantially when reacting amino groups were not available. A mechanism of EDC-induced gelatin degradation is proposed and designated the extended Khorana mechanism. This EDC side reaction has the potential to occur in peptides and proteins under similar conditions.