title: Carbodiimide induced cross-linking, ligand addition, and degradation in gelatin.
abstract: 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.
authors: Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia , 600 South 43rd Street, Philadelphia, Pennsylvania 19104-4495, United States.
authors: Christopher R
authors: CR
authors: Cammarata
authors: Mitchell E
authors: ME
authors: Hughes
authors: Clyde M
authors: CM
authors: Ofner
mesh_terms: D000818:Animals; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D005780:Gelatin / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008956:Models, Chemical; D008970:Molecular Weight; D059748:Proteolysis; D014867:Water
chemical_list: D002021:Buffers; D003432:Cross-Linking Reagents; D006834:Hydrazines; D008024:Ligands; D014867:Water; C029424:hydrazine; D005780:Gelatin; D005022:Ethyldimethylaminopropyl Carbodiimide
keywords: amide bond formation; carbodiimide; carboxylic anyhydride; cross-linking; degradation; gelatin