Detection of subtle differences in the surface structure of lysozymes by use of an immobilized Fab fragment

Tadashi Ueda, Yoshito Abe, Kenji Akasaki, Yasunori Yamaguchi, Hiroshi Tsuji, Keiichl Kawano, Hidenori Yamada, Taiji Imoto

Research output: Contribution to journalArticle

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Abstract

A method was developed to evaluate the association constant at physiological pH (pH 7.5) between a lysozyme and the Fab fragment derived from anti-lysozyme monoclonal antibody 37-7, which was immobilized to the adsorbent for HPLC. Comparison of the association constants between lysozymes and the immobilized Fab fragment indicated that mAb 37-7 recognized the prominently exposed regions (hills and ridges) around His15 of hen lysozyme, but HislS itself was not directly involved in the binding with mAb 37-7. Moreover, the epitope was confirmed by the reactivity of HislS with monoiodoacetic acid in the presence of mAb 37-7. The association constant of 15-carboxymethylated histidine lysozyme (15CM lysozyme) with the immobilized Fab fragment was smaller by oneseventh than that of 15-carboxamidated histidine lysozyme, though the side chains introduced were almost identical in size. From the pH titration of 15CM lysozyme with 13C-enriched carboxyl group by use of 13C-NMR, the pJfa of the introduced carboxyl group was evaluated to be 5.06. Since the carboxyl group was fully ionized under the conditions of measurement (pH 7.5), electrostatic repulsion was found to disturb severely the association between mAb 37-7 and hen lysozyme. Moreover, it was demonstrated that, because of the high reproducibility of measurement, the immobilized Fab fragment could detect subtle differences in the surface structure of lysozymes. 1993

Original languageEnglish
Pages (from-to)174-179
Number of pages6
JournalJournal of biochemistry
Volume113
Issue number2
DOIs
Publication statusPublished - Jan 1 1993
Externally publishedYes

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Immunoglobulin Fab Fragments
Muramidase
Surface structure
Histidine
Association reactions
Iodoacetic Acid
Static Electricity
Titration
Adsorbents
Epitopes
Electrostatics
High Pressure Liquid Chromatography
Monoclonal Antibodies
Nuclear magnetic resonance

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Detection of subtle differences in the surface structure of lysozymes by use of an immobilized Fab fragment. / Ueda, Tadashi; Abe, Yoshito; Akasaki, Kenji; Yamaguchi, Yasunori; Tsuji, Hiroshi; Kawano, Keiichl; Yamada, Hidenori; Imoto, Taiji.

In: Journal of biochemistry, Vol. 113, No. 2, 01.01.1993, p. 174-179.

Research output: Contribution to journalArticle

Ueda, Tadashi ; Abe, Yoshito ; Akasaki, Kenji ; Yamaguchi, Yasunori ; Tsuji, Hiroshi ; Kawano, Keiichl ; Yamada, Hidenori ; Imoto, Taiji. / Detection of subtle differences in the surface structure of lysozymes by use of an immobilized Fab fragment. In: Journal of biochemistry. 1993 ; Vol. 113, No. 2. pp. 174-179.
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