Determination of free N-acetylamino acids in biological samples and N-terminal acetylamino acids of proteins

N-Acetylamino acids were derivatized with 9-anthryldiazomethane to the corresponding esters. The anthryl esters were separated by high-performance liquid chromatography and detected fluorimetrically (excitation at 365 nm; fluorescence emission measured at 412 nm). N-Acetyl derivatives of Asn, Gln, Ser, Thr, Gly, Ala, Tyr, Pro, Met, Val, Ile and Leu as well as N-formyl-Met could be separated and identified in the same chromatographic run. The detection limit was from 0.10 pmol for AcGln to 5.5 pmol for AcIle and AcLeu. When the acetylamino acids listed above were added to the 700 g supernatant of a rat liver homogenate, the mean recovery was 72%. AcAla and AcTyr were found in free form in baker's yeast. Proteins with an acetylated N-terminus were digested by a protease, and the peptides formed were treated with an N-acylamino acid-releasing enzyme. This method was applied to end-group determination of four proteins (each 0.5 nmol).