β-Galactoside-binding activity of human galectin-1 (hGal-1) was evaluated at pH 7-9.5 by fluorescence spectroscopy from the fraction bound to lactose gel (Y) and the lactose binding constant (Kb). Y decreases at pH > 8.2 ± 0.1 in the absence of NaCl, while it is constant in the presence of 150 mM NaCl. On the other hand, Kb is independent of pH and the NaCl concentration at basic pH. Analysis of Raman spectrum has shown that the pKa of Cys residues of hGal-1 is 8.5 ± 0.1 on average, indicating that about 40% of the six Cys residues of hGal-1 would be deprotonated at pH 8.2. The pH dependence of Y is explained by an increase of Coulombic repulsion among negatively charged hGal-1 on the lactose gel surface. This result suggests that Y is not always a good indicator of the β-galactoside-binding activity of galectins, which contain many Cys residues.
ASJC Scopus subject areas