Solid‐Phase Edman Degradation

A modification of the Edman degradation is described whereby peptides are degraded sequentially on a solid support by a machine, the solid‐phase peptide sequencer. Reagents are pumped through a column containing the resin‐bound peptide, and the amino acid thiazolinones released after each degradative cycle are collected on a fraction collector for subsequent analysis. A complete cycle requires 2 h, and all operations are controlled automatically by a programmer. An improved peptide resin is described, to which peptides can be attached in yields ranging from about 70% for the 30 amino acid insulin B‐chain to nearly 100% for smaller peptides. Of the 18 amino acids examined so far, glycine, alanine, phenylalanine, tyrosine, valine, leucine, isoleucine, proline, methionine, asparagine, glutamine, histidine, lysine, threonine, serine and cysteic acid are degraded normally; the phenylthiohydantion of threonine is partially dehydrated and that of serine is almost completely destroyed by trifluoroacetic acid, however. The phenylthiohydantion of glutamic acid is not detected, because it remains bound by its γ‐carboxyl group to the resin. When aspartic acid is encountered in peptides, further degradation is completely blocked, presumably as a result of the β‐carboxyl group having formed a cyclic imide. Using the sequencer and unpurified reagents, it was possible to degrade entirely a 0.24 μmole sample of the 21 amino acid insulin A‐chain (oxidized) and to analyze the products within a 3‐day period. Degradative yields averaged 94% per cycle. Similarly, 0.1 μmole of the insulin B‐chain was degraded through 18 cycles before identification of the phenylthiohydantoins became ambiguous. The sequencer is simple in design and operation, and is inexpensive to construct. The scope and limitations of the method are discussed.