The consensus octapeptide repeat motif of the barley seed storage
protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the
epitope of two anti-prolamin
monoclonal antibodies (
Mabs), IFRN 0061 and 0614. The
Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C
hordein and a
peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by
enzyme-linked immunosorbent assay (
ELISA). The K(d) of IFRN 0614 for the consensus
peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar
ELISA binding characteristics were observed with an anti-C
hordein polyclonal antiserum and a
Mab raised to the consensus
peptide.
Circular dichroism (CD) and Fourier-transform
infrared (FTIR)
spectroscopy showed that the protein and the consensus
peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and
beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce
antibody binding at higher temperatures, they cannot account for the complete loss of
Mab recognition at higher temperatures. It seems likely that the
Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.
