AUTOMATED EDMAN DEGRADATIONS - STUDIES WITH A LARGE SEQUENCER CUP AND HIGH-SPEED DRIVE

The major limitations with the current methodology employed in the automated Edman degradation of polypeptides and proteins is the progressive increase in overlap of penultimate amino acid residues during degradation and the appearance of new amino-terminal amino acid residues resulting from nonspecific cleavage of peptide bonds during acid cleavage. These limitations have been significantly reduced with automated degradations performed on a modified Beckman 890B sequencer equipped with a large reaction cup (75% increase in surface area), high speed drive (1800/3600 rpm), and cold trap attached to the high vacuum pump. Automated Edman degradations performed with the modified Beckman sequencer on apomyoglobin and bovine parathyroid hormone were of higher repetitive yield, and the individual steps in the sequence contained significantly less overlap and nonspecific background of amino acids due to acidolysis. Degradations with the modified sequencer on apomyoglobin were routinely performed for 65-70 cycles, and 2 to 20 mg of protein was efficiently sequenced with the large reaction cup. With the new modified sequencer, a single degradation can be extended further down the protein chain, and large as well as small proteins can be degraded. This improved system will facilitate the structural analysis of polypeptides and proteins and will reduce the necessity for extensive cleavage of proteins for overlapping peptide fragments. © 1979 Academic Press, Inc.