The modification and removal of the N-terminal residue of trypsin by transamination.

Dixon (1964a,b) showed that peptides could be transaminated non-enzymically, with conversion of the N-terminal amino acid residue into an oc-oxoacyl residue. Conditions can be selected that are sufficiently mild to offer the hope that the biological activity might be retained in many cases. The method has been applied successfully to cytochrome c-551 by Dixon & Moret (1964), to carboxypeptidase by van Heyningen et al. (1968) and to insulin by Bunzli & Bosshard (1971). The x-oxoacyl residue can be cleaved selectively from the rest of the molecule by treatment with a bifunctional nucleophile, o-phenylenediamine (Dixon & Moret, 1964; van Heyningen & Dixon, 1967). Although it is possible to modify and remove the N-terminal residue of a protein by the use of the Edman (phenyl isothiocyanate) procedure with retention of activity [e.g. for insulin (Brandenburg, 1969; Borras & Offord, 1970)], transamination, which is more mild and is specific for a-amino groups, is probably of more general application. We believe, in fact, that the method merits wider use, and the desire to demonstrate its successful application in the particularly unfavourable circumstance of a protein with N-terminal isoleucine (see below) was one reason for undertaking this work. In particular we wished to explore the transamination procedure as a means of bringing about temporary protection of the N-terminus of the chain. It is then possible to protect the E-amino groups, before exposing an a-amino group again, at which peptide couplings can be carried out with the production of semisynthetic proteins. We find that transamination is quite suitable for this purpose. An unexpected finding was that, in the course ofthe conversion ofthe original a-amino group into an oxo group, the protein becomes extremely insoluble. This must presumably be as a result of a conformational change, which we infer, on the basis of the great decrease in solubility, to be of considerable magnitude. This change is particularly striking when compared with the rather minor modification that brought it about.