Influence of ions on cyclization of the amino terminal glutamine residues of tryptic peptides of streptococcal PepM49 protein. Resolution of cyclized peptides by HPLC and characterization by mass spectrometry.
RPHPLC of the tryptic digest of lysine blocked group A streptococcal PepM49 protein (DHP-PepM49) consistently yielded, among others, two pairs of peptides which were well resolved, eluted in tandem, and had identical amino acid compositions. In each pair, the earlier eluting peptide was readily amenable to sequencing and yielded an amino-terminal glutamine whereas the later eluting peptide could not be sequenced. Mass spectral analysis revealed that each of these pairs of peptides differed in mass corresponding to the loss of ammonia. These data suggested that the later eluting peptide in each pair is a result of cyclization of the amino-terminal glutamine residue to pyroglutamic acid, which apparently leads to an increase in the hydrophobicity of the peptide. A kinetic analysis of the tryptic digestion of the DHP-PepM49 protein revealed that the cyclized form of the peptides were essentially absent during the initial time and increased with time of incubation, with a concomitant decrease in the uncyclized form. In 0.2 M ammonium bicarbonate at 37 degrees, nearly 44% conversion of the glutaminyl peptides to the pyroglutamyl peptides was observed in 24 h. This conversion was accelerated in sodium phosphate buffer relative to that in ammonium bicarbonate whereas it had a significantly lower rate in ammonium acetate buffer. The conversion was also temperature dependent, with essentially no cyclization at 0 degree, in all the three buffers. Thus, an extended digestion at 0 degree or a brief digestion at 37 degrees in ammonium acetate was found to be a suitable condition for limiting the cyclization of amino-terminal glutamine residues of PepM49 peptides.(ABSTRACT TRUNCATED AT 250 WORDS)
Khandke
K M
KM
Rockefeller University, New York, NY.
Fairwell
T
T
Chait
B T
BT
Manjula
B N
BN
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002851:Chromatography, High Pressure Liquid; D005973:Glutamine; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D014357:Trypsin
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D010446:Peptide Fragments; D010456:Peptides, Cyclic; C014689:streptococcal M protein; D005973:Glutamine; D014357:Trypsin