Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies.

David M Kranz, J. N. Herron, E. W. Voss

Research output: Contribution to journalArticle

Abstract

Binding of fluorescyl ligand by five IgG anti-fluorescyl hybridoma proteins (4-4-20, 6-10-6, 20-4-4, 20-19-=1, 20-20-3) was examined. Relative reduction in fluorescence of bound fluorescein, deuterium oxide (D2O)-induced enhancement of fluorescence, and the effects of pH on binding kinetics were measured for each clone. Individual hybridoma proteins (all of which bind fluorescein with relatively high affinity) exhibited significant differences in the relative contribution of various forces (hydrophobicity, hydrogen bonding, ionic interactions) to binding and hence, affinity. The extent of such variations in binding mechanisms among monoclonal antibodies binding the same hapten is indicative of the extreme functional diversity of active sites. In addition, ligand binding by clone 20-20-3 was examined in greater detail. ABsorption spectra of ligand bound by purified intact antibody, Fab fragments, and reassociated heavy and light chains indicated that protonation of the fluorescyl ligand by a residue within the active site contributed significantly to the binding free energy. Comparative dissociation rates of fluorescein and a structural analog, rhodamine 110, were used to quantitatively substantiate the contribution of this interaction. Association and dissociation rate studies with fluorescein and antibody indicated that: 1) the active site appeared to undergo a conformational change upon ligand binding, and 2) neither intact disulfides nor intersite cooperativity affected the dissociation rate of bound ligand. Observed mechanisms of ligand binding are discussed in terms of proposed mechanisms of antibody affinity maturation and diversity.

Original languageEnglish (US)
Pages (from-to)6987-6995
Number of pages9
JournalJournal of Biological Chemistry
Volume257
Issue number12
StatePublished - Jun 25 1982
Externally publishedYes

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Anti-Idiotypic Antibodies
Monoclonal Antibodies
Ligands
Fluorescein
Catalytic Domain
Hybridomas
Antibodies
Antibody Diversity
Clone Cells
Fluorescence
Deuterium Oxide
Immunoglobulin Fragments
Immunoglobulin Fab Fragments
Antibody Affinity
Haptens
Protonation
Hydrogen Bonding
Hydrophobicity
Hydrophobic and Hydrophilic Interactions
Disulfides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies. / Kranz, David M; Herron, J. N.; Voss, E. W.

In: Journal of Biological Chemistry, Vol. 257, No. 12, 25.06.1982, p. 6987-6995.

Research output: Contribution to journalArticle

Kranz, David M ; Herron, J. N. ; Voss, E. W. / Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies. In: Journal of Biological Chemistry. 1982 ; Vol. 257, No. 12. pp. 6987-6995.
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