Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten

Elisar Barbar, Tammy Martin, McKay Brown, Marvin Rittenberg, David H. Peyton

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The structural basis of the binding of phenylphosphocholine haptens to antibodies was studied. This was done by preparing antibodies and testing binding to conjugates of phenylphosphocholine. The choice of haptens was made in order to evaluate the contribution of the carrier to binding, and its effect on hapten conformation in the active site. Thus, phosphocholine (PC) was diazophenyl-linked to tyrosine or histidine as single amino acid carriers and to tripeptides or octapeptides containing tyrosine or histidine as central amino acids to which PC was attached. Relative affinity was assessed by inhibition enzyme-linked immunosorbent assay (ELISA) and binding constants were determined by fluorescence quenching. Fluorinated haptens were used to determine the kinetics of binding using 19F nuclear magnetic resonance. The transferred nuclear Overhauser effect was used to characterize conformation of the bound hapten. We had previously shown that nitrophenylphosphocholine unlinked to carrier is bound in the active site as a bent structure [Bruderer, U., Peyton, D. H., Barbar, E., Fellman, J. H., and Rittenberg, M. B. (1992) Biochemistry 31, 584-589]. We show here that this same bent conformation is retained in the active site regardless of the neighboring carrier or the conformation of the hapten in the unbound conjugate. The presence of the carrier residues in the bound state does, however, influence affinity.

Original languageEnglish (US)
Pages (from-to)2958-2967
Number of pages10
JournalBiochemistry
Volume35
Issue number9
DOIs
StatePublished - Mar 5 1996

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Haptens
Conformations
Binding Sites
Antibodies
Catalytic Domain
Phosphorylcholine
Histidine
Tyrosine
Enzyme inhibition
Amino Acids
Immunosorbents
Biochemistry
Quenching
Assays
Magnetic Resonance Spectroscopy
Fluorescence
Enzyme-Linked Immunosorbent Assay
Nuclear magnetic resonance
Kinetics
Testing

ASJC Scopus subject areas

  • Biochemistry

Cite this

Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten. / Barbar, Elisar; Martin, Tammy; Brown, McKay; Rittenberg, Marvin; Peyton, David H.

In: Biochemistry, Vol. 35, No. 9, 05.03.1996, p. 2958-2967.

Research output: Contribution to journalArticle

Barbar, Elisar ; Martin, Tammy ; Brown, McKay ; Rittenberg, Marvin ; Peyton, David H. / Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten. In: Biochemistry. 1996 ; Vol. 35, No. 9. pp. 2958-2967.
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