Characterization of Covalent Protein Conjugates Using Solid-State ¹³C NMR Spectroscopy

Cross-polarization magic-angle spinning (CPMAS) ¹³C NMR spectroscopy has been used to characterize covalent conjugates of alachlor, an α-chloroacetamide hapten, with glutathione (GSH) and bovine serum albumin (BSA). The solid-state NMR method demonstrates definitively the covalent nature of these conjugates and can also be used to characterize the sites of hapten attachment to proteins. Three different sites of alachlor binding are observed in the BSA system. Accurate quantitation of the amount of hapten covalently bound to GSH and BSA is reported. The solid-state ¹³C NMR technique can easily be generalized to study other small molecule/protein conjugates and can be used to assist the development and refinement of synthetic methods needed for the successful formation of such protein alkylation products.