Practical Protein Chemistry - A. Darbre 1989

Determination of C-terminal amino acid sequence
Determination of C-terminal groups
Selective reduction

Chemical reduction of the C-terminal amino acid to the corresponding alcohol was the first chemical reaction used to determine the C-terminal amino acid of Proteins. For instance, Insulin was heated with LiAlH₄ in N-ethylmorpholine at 55 °C for 8 h, and the resulting amino alcohols were identified by paper Chromatography [26]. The reduction reaction was also carried out after preliminary Esterification of all protein carboxyl groups [17]. In this Procedure, the mild reducing agent LiBH₄ in tetrahydrofuran was used, followed by the Quantitative determination of the amino alcohols derived from the C-terminal Amino Acids. Alongside the main processes, reductive Cleavage of peptide bonds (1–2%) and conversion of peptide carbonyls into methylene groups also take place.

Commercially available sodium dihydro-bis(2-methoxyethoxy)aluminate was used to reduce the C-terminal amino acids of di- and tripeptides [75]. This method eliminates The Need for preliminary esterification of the carboxyl groups. The amino alcohols were identified by paper chromatography. To date, there are no reports on the application of this reduction METHOD FOR DETERMINING the C-terminal amino acids of proteins. It has been reported that NaBH₄ in aqueous solution (but not in an organic solvent) selectively and almost quantitatively reduces esterified C-terminal amino acids to their corresponding alcohols [32]. When working with Lysozyme, bovine insulin, and concanavalin A, the expected derivatives were obtained without any Side Reactions being detected. This approach could be developed into a practical technique for C-terminal protein analysis, although its further advancement largely depends on the reliability of the analytical Determination of the amino alcohols derived from all common amino acids.