A method for determining the free amino groups on the surface of proteins by capillary electrophoresis is described. A protein solution was equally divided into 5 vials. Different amount of excess 9-fluorenylmethoxycarbonylsuccinimide (FMOC-OSu) solution was then added to each vial, followed by mixing and keeping at room temperature for 30 min. The unreacted FMOC-OSu was separated from the protein by a micellar electrokinetic chromatography (MEKC). The peak area of the unreacted FMOC-OSu was measure and the calibration curve was established between the molar numbers of added FMOC-OSu against the corresponding peak areas of unreacted FMOC-OSu. The molar numbers of amino groups on the surface of protein should be equal to that of the reacted FMOC-OSu, which could be gained by extending the calibration curve to cross the x-axis, where the peak area of unreacted FMOC-OSu was zero implying that all added FMOC-OSu reacted with the amino groups. Glycine solution was used to validate the method. Bovine serum albumin (BSA) was selected as a model protein and the number of free amino groups on the surface of the BSA was determined as 32, which is consistent with the reported value. The method can be potentially used to determine the average modification degree of the PEGylation proteins.

Key words: free amino groups of protein, determination of the amino group, micellar electrokinetic chromatography