A RAPID HPLC DETERMINATION OF THE ISOMERIZATION LEVEL OF ASP-RESIDUES WITHIN SYNTHETICα-A-CRYSTALLIN FRAGMENTS

Determination of rate constants for β-linkage isomerization of three specific aspartyl residues in recombinant human αA-crystallin protein by reversed-phase HPLC

“…Because the isoAsp/Asp ratio in the T6 peptide [20] and model hexapeptide [10] was shown to reach 3.6 at equilibrium state, we assume k: k = 3.6: 1. Thus, Eq.…”

“…The incubated peptide samples were analyzed by HPLC using a JASCO HPLC system 880 (Nippon Bunko Co., Ltd); a C18 column, Develosil ODS-UG-5 (4.6 mm id × 150 mm length, Nomura Chemical Co., Ltd); or Capcell Pak C18 UG120 (4.6 mm id × 150 mm length, Shiseido Co., Ltd). The separation of T6 and T18 peptides was carried out according our previous study [20,21]. The separation of the T6, T10, and T18 peptides was carried out using the mobile phases of acetonitrile-sodium phosphate solutions (pH 5.1, 15 mM (15:85, v/v); pH 3.0, 15 mM (18:82, v/v); and pH 3.0, 15 mM (11:89, v/v); respectively).…”

“…We developed a simple method for quantifying the structural alterations of the 58 Asp, 84 Asp, and 151 Asp residues in human ␣A-crystallin protein using RP-HPLC with a standard octadecylsilane (ODS) column by referring to our previous studies [20,21] because the traditional method for the quantification of ␤-linked Asp residues was time-consuming and not sensitive. Kinetic studies revealed that the ␤-linkage isomerization of the 58 Asp residue was suppressed in the recombinant protein (by approximately 0.4-0.5 times compared to Asp isomerization in the synthetic peptide), while the isomerization of the 151 Asp residue occurred at the same rate as that in the synthetic peptide.…”

Quantification of Structural Alterations of L‐Asp and L‐Asn Residues in Peptides Related to Neuronal Diseases by Reversed‐Phase High‐Performance Liquid Chromatography

“…In this study, the separation condition for analyzing peptide isomers related to the prion disease and Alzheimers disease can be determined by adjusting the pH and salt concentration of the mobile phase. We have succeeded in the separation of different types of peptide isomers [10] [11]. The results of these separations reveal that the structural alterations in different types of peptide isomers are quickly quantified by RP-HPLC with the mobile phase containing a phosphate solution and an appropriate concentration of NaCl; the results also suggest that the present method can be universally used for the analysis of the structural alterations of Asn and Asp in peptides.…”

“…The analyses of structural alterations in Asp residues have been successfully carried out in different types of peptides including the three peptide fragments of human aA-crystallin, 304 (The underlined Asp residues vary with the structurally altered Asp.) [10] [11].…”