The pH stability of lectin from black turtle bean was characterized by a variety of biophysical
techniques over the pH range from 2.0 to 10.0. The zero-order and second-derivative UV spectra at different
pH values indicated that the tertiary structure of the protein does not change significantly with changes
in pH. The maxima emission fluorescence of the lectin upon excitation at 280 nm and 295 nm were found
to be blue shifted 1.2 nm at pH 2.0, and with a red-shift of 1.0 nm at pH 10.0. The chemical denaturation
results obtained by monitoring the intrinsic fluorescence of the lectin indicated that unfolding of the protein
induced by guanidine hydrochloride (GdnHCl) could be described using a three-state model. Complete
unfolding of the protein was observed at pH 7.2 in the presence of 6.5 M GdnHCl after 2 weeks,
which suggested that the lectin was stable at various pHs. Irreversible thermal denaturation of lectin was
also investigated at various pHs by differential scanning calorimetry (DSC). The first-order two-state kinetic model was
applied to explain the scan-rate dependent DSC transitions. Both the higher activation energy (Ea) computed from the irreversible
thermal denaturation and the three-state chemical denaturation process suggested that the lectin structure remained
significantly unchanged from pH 2.0 to 10.0, which indicated that the structure of the lectin was stable over a
wide pH range.
Keywords: Black turtle bean, differential scanning calorimetry, fluorescence spectroscopy, lectin, stability, UV spectroscopy.
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