Steady State and Time-Resolved Fluorescence Studies of a Hemagglutinin from Moringa oleifera
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The saccharide binding and conformational characterization of a hemagglutinin, a low molecular weight protein from the seeds of Moringa oleifera was studied using steady state and time resolved fluorescence. The lectin binds sugars LacNAc (K a = 1380 M−1) and fructose (K a = 975 M−1), as determined by the fluorescence spectroscopy. It has a single tryptophan per monomer which is exposed on the surface and is in a strong electropositive environment as revealed by quenching with iodide. Quenching of the fluorescence by acrylamide involved both static (K s = 0.216 M−1) and collisional (K sv = 8.19 M−1) components. The native protein showed two different lifetimes, τ 1 (1.6 ns) and τ 2 (4.36 ns) which decrease and get converted into a single one, (2.21 ns) after quenching with 0.15 M acrylamide. The bimolecular quenching constant, k q was 7.55 × 1011 M−1 s−1. ANS binding studies showed that the native protein has exposed hydrophobic patches which get further exposed at extreme acidic or alkaline pH. However, they get buried in the interior of the protein in presence of 1 M GdnHCl or urea.
KeywordsMoringa oleifera Hemagglutinin Saccharide binding ANS binding Fluorescence Solute quenching Lifetime
The authors wish to thank Dr. M. B. Badiger for permission to use spectrofluorimeter. UVK thanks CSIR for a research fellowship.
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