Determination of the N-terminal Amino-acid in Polypeptides and Proteins

Abstract

THE weakest point in the DNP-method¹ is the destruction of the N-terminal DNP-amino-acid which occurs under the conditions of hydrolysis necessary to detach it from the protein. The optimum time of hydrolysis for an end-group analysis is the time at which DNP-peptides are no longer present; this is different with every protein, but overnight hydrolysis (16–24 h) with constant boiling HCl is routine. The extent of destruction is different with each protein, and is generally much greater than would be expected for any free DNP-amino-acid under the same conditions of hydrolysis in the absence of protein. It is not possible to correct for destruction by hydrolysis for a graded series of times and extrapolation to zero time, for much of the destruction seems to take place during the initial hours of the hydrolysis before degradation has proceeded to small fragments. Thus, although the DNP-method has been, and will doubtless continue to be, widely used for end-group analysis, it is slowly being superseded by stepwise degradation methods.