Pyroglutamate aminopeptidase

Reactions of Hez-PBAN. A dried aliquot of purified Hez-PBAN was allowed to react with pyroglutamate aminopeptidase as described previously (4). The quenched reaction mbcture was analyzed under the conditions of RP-HPLC Step D. A dried aliquot of purified synthetic Hez-PBAN was oxidized by reaction with DMSO/HCl/acetic acid, as described previously (1). The major product was purified by the conditions of RP-HPLC Step D. The purified Hez-PBAN disulfoxide was subsequently sequenced on the Model 477A sequencer. 

Moyer, M., A. Harper, G. Payne, H. Ryals and E. Fowler. In situ digestion with pyroglutamate aminopeptidase for N-terminal sequencing of electroblotted proteins. J. Protein Chem. 9 282—283, 1990. 

Figure 4.106 Separation of blocked and W-1-peptides after treatment with pyroglutamate aminopeptidase. Separator column ProPac WCX-10 eluent (A) 0.02 mol/L sodium phosphate (pH 4.8) and (B) 0.02 mol/L sodium phosphate +1 mol/L NaCI (pH 4.8) gradient linear, 0% b to 60% B in 15 min flow rate ...

Another example of high resolution of tentacular cation exchangers is the separation of peptides with Af-terminal Glu- or Gin residues that tend to imdergo cyclization reactions, by which the /V-terminal end will be blocked. This blocked residue can be specifically removed with pyroglutamate aminopeptidase. As can be seen from Figure 4.106, the ProPac WCX-10 weak acid cation exchanger is suitable for the separation of blocked peptides from the AM-peptides. 

Fig. 3-219. Separation of blocked and N-l-peptides after treatment with pyroglutamate aminopeptidase. - Separator column ProPac WCX-10 eluant (A) 0.02 mol/L sodium phosphate, pH 4.8, (B) 0.02 mol/L sodium phosphate, pH 4.8 + 1 mol/L Nad gradient linear, 0% B to 60% B in 15 min flow rate 1 mL/min detection UV (220 nm) samples A. pGlu-Ser-Leu-Arg-Trp-amide 4- pyroglutamate aminopeptidase, B. pGlu-Ser-Leu-Arg-Trp-amide, C. neurotensin -i- pyroglutamate aminopeptidase, D. neurotensin (pGlu-Leu-Tyr-Gln-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu), E. buffer-i-enzyme control (0.1 mmol/L sodium phosphate, lOmmol/L Na2-EDTA, 5 mmol/L DTT, 5% (v/v) glycerol, pH 8.

---