Quantification of the isomerization of Asp residue in recombinant human alpha A-crystallin by reversed-phase HPLC.
A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp(151) in recombinant human alphaA-crystallin. First, four types of dodecapeptide fragment ((146)IQTGLD(151)ATHAER(157)) in which the Asp residue was either L-Asp, D-Asp, L-isoAsp or D-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp(151)-containing peptide fragments isolated from the tryptic hydrolysate of recombinant alphaA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing D-Asp, L-isoAsp and D-isoAsp, were generated when alphaA-crystallin was incubated for 140 days at 37 degrees C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing L-isoAsp and D-isoAsp represented dodecapeptide fragments containing L-isoAsp(151) and D-isoAsp(151), respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing D-Asp represented dodecapeptide fragments containing D-Asp(151). The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp(151) residue in human alphaA-crystallin, the degree of isomerization from L-Asp to D-Asp can be determined to a level as low as 0.3%.
Sadakane
Yutaka
Y
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. sadapon@toyama-mpu.ac.jp
Yamazaki
Toshiaki
T
Nakagomi
Kazuya
K
Akizawa
Toshifumi
T
Fujii
Noriko
N
Tanimura
Takenori
T
Kaneda
Masaki
M
Hatanaka
Yasumaru
Y
D001224:Aspartic Acid / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis*; D013237:Stereoisomerism; D038202:alpha-Crystallin A Chain / Q000032:analysis*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D001224:Aspartic Acid