Deblocking of Proteins Containing N-Terminal PyroglutamicAcid

Abstract

Pyroglutamic acid (pGlu) is the product of cyclization of N-terminal glutamine and is one of the common modifications found at the N-termini of proteins. This modification is typical for immunoglobulins, especially IgGs, since they contain glutamine at the amino terminus of the heavy chain. This chemical modification, in addition to other modifications of the N-terminus results in a “blocked“ protein which cannot be sequenced using Edman degradation. Many deblocking procedures have been reported to remove common modifications at the N-terminal end of proteins for the sequence determination by Edman chemistry (1). Deblocking of N-terminal pGlu is usually achieved by the use of either chemical methods (2,3) or by the use of pyroglutamate aminopeptidase (PGAP) (1,4,5). Removal of pGlu from peptides by PGAP with high yield has been described (1,6–10) and recommended as a standard procedure (1). However, when this protocol is applied to large proteins such as IgGs, it results in relatively low yield (<40%) of the deblocked protein. The most commonly cited procedure of Podell and Abraham (11) for removal of pGlu from proteins is also recommended in the handbooks (1,12). However, this procedure is not consistent with the results, which demonstrated that incubation at 4°C did not yield any deblocking (13). Other existing protocols use PGAP for digestion in solution or for proteins blotted onto PVDF membranes and result in variable yields of deblocked protein (2,11,12,15,16).