Evaluation of the conformational equilibrium of reduced hen egg lysozyme by antibodies to the native form

Masayuki Oda, Aki Kitai, Akikazu Murakami, Miyuki Nishimura, Takatoshi Ohkuri, Yoshito Abe, Tadashi Ueda, Haruki Nakamura, Takachika Azuma

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

To evaluate the conformation of reduced HEL, the monoclonal antibodies HyC1 and HyC2, which recognize different conformational epitopes on native hen egg lysozyme (HEL), were used, and the kinetics of their interactions with native HEL, S-1,2-dicarboxyethylated HEL (DCE-HEL), and carboxymethylated Cys6 and Cys127 HEL (CM6,127-HEL) were assessed using surface plasmon resonance. Although their association rate constants differed 105-fold, their dissociation rate constants were essentially the same, suggesting that DCE-HEL and CM6,127-HEL possess conformations similar to that of native HEL when they bind antibodies. We considered that the ratio of the association rate constant of reduced HEL to native HEL represents the proportion of the native format determinant in equilibrium. Reduction of the Cys6-Cys127 disulfide bond would transform the epitope recognized by HyC1 into a non-native conformation similar to that of DCE-HEL. We show that monoclonal antibodies provide a sensitive tool for evaluation of the structural and hydrodynamic changes of proteins.

Original languageEnglish
Pages (from-to)145-150
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume494
Issue number2
DOIs
Publication statusPublished - Feb 2010

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

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