ACTIVATION OF THE CARBOXY-TERMINUS OF A PEPTIDE FOR CARBOXY-TERMINAL SEQUENCING

Previously, we reported [Anal. Biochem. 1992, 206, 344-352] a new meth od of sequencing proteins from the carboxy terminus (C-terminus). The carboxyl group at the C-terminus is activated and derivatized into a t hiohydantoin (TH). We reported that, by alkylating the TH formed at th e C-terminus, the TH is converted into a readily displaced leaving gro up. Reaction with {NCS}(-) under acidic conditions cleaves the alkylat ed thiohydantoin (ATH) and derivatizes the freshly exposed C-terminus into a new proteinyl-TH. The efficiency of the initial activation of t he carboxy group at the C-terminus is critical to the initial yield of the first ATH residue. In order to directly observe the intermediates that form during activation of the C-terminus, a model tripeptide, ac etylalanine-alanine-alanine-OH (Ac-Ala-Ala-Ala-OH) was subjected to th e reagents used to form the peptidyl-TH, Ac-Ala-Ala-Ala-TH. The reacti on was monitored by nuclear magnetic resonance spectroscopy. An oxazol one was observed to form immediately at the C-terminus during the reac tion with diphenyl chlorophosphate (DPCP), tetraphenyl pyrophosphate ( TPPP), or tetramethylchlorouronium chloride (TMU-Cl). The oxazolone wa s observed to react with an excess of the carboxy group-activating rea gents while under basic conditions. Diketopiperazine formation at the C-terminus was also observed. These side reactions prevent or retard t he reaction of (NCS)- to form a peptidyl-TH and correlate with a reduc ed initial yield observed during automated C-terminal protein sequenci ng. The carboxylic acid-reactive reagents react with the side-chain ca rboxylic acid groups of aspartic and glutamic acid residues as well as the C-terminus. We found that the sidechain carboxylic acid groups in a protein could be selectively amidated in the presence of the protei nyl-oxazolone at the C-terminus.