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A highly sensitive lectin-based impedimetric detection method has been developed for the determination of galactose in human serum samples. The present method is based on the selective interaction between a target galactose and a galactose-binding lectin, Ricinus communis agglutinin I (RCA120). The quantitative detection of galactose in human serum samples relies on the competition reaction of the target galactose of interest with β-galactose derivative immobilized on a gold electrode surface for a limited amount of RCA120. At equilibrium, the amount of bound RCA120-β-galactose complex on gold electrode surface is inversely proportional to the amount of the target galactose in sample solution. The binding of RCA120 onto the β-galactose-immobilized gold electrode was monitored through electron transfer resistance, which was directly measured by electrochemical impedance spectroscopy. The present method can detect galactose from 8.33 × 10−9 M to 1.25 × 10−6 M (r2 = 0.992) with a limit of detection of 5.87 × 10−9 M (S/N = 3), which is much lower than those obtained with enzyme-based amperometric and colorimetric detection methods. The present method was successfully applied to the determination of galactose in human serum samples.

Acid denaturation of champedak galactose-binding (CGB) lectin was studied in the pH range, 7.0-1.0 using intrinsic fluorescence and ANS fluorescence measurements. The lectin remained stable up to pH 5.0 and showed local disordering in the vicinity of the protein fluorophores within the pH range, 5.0-3.5. Decrease in the pH from pH 3.5 to pH 2.5 led to structural transition, marked by the decrease in the intrinsic fluorescence and increase in the ANS fluorescence signals. This can be ascribed to the dissociation of the tetrameric lectin into monomeric forms. Further decrease in the pH up to pH 1.5 produced another transition, which specified the unfolding of monomers as reflected from the decrease in both intrinsic fluorescence and ANS fluorescence signals. Characterization of the conformational states obtained at pH 7.0, pH 2.5 and pH 1.5 based on intrinsic and ANS fluorescence spectra, gel chromatographic behavior and thermal denaturation confirmed the existence of folded monomeric forms at pH 2.5 and unfolded states at pH 1.5. However, the aciddenatured state of CGB lectin at pH 1.5 retained significant residual structure, as evident from the greater loss of both secondary and tertiary structures in the presence of 6 M guanidine hydrochloride at low pH values. Anion-induced refolding below pH 1.5 was also seen using ANS fluorescence measurements.

It had previously been found that small unilamellar liposomes of ca. 0.03 μm diameter which bear synthetic cholesterol-containing glycolipids may be aggregated by an appropriate lectin [8]. Where studied, threshold effects have been observed in that the amount of glycolipid incorporated in the liposomes must exceed a certain minimum concentration in order for aggregation to occur [3, 8, 9, 13, 14]. Threshold effects of this type may be important in mediating cell-cell and virus-cell interactions. However, before studies with small unilamellar liposomes are useful as a model for these recognition and binding phenomena, it must be shown that the observed threshold effects are not associated with the very small radius of curvature of these liposomes. This article reports that larger liposomes of average diameter 0.26 and 0.45 μm which contain the synthetic glycolipidl also show threshold effects when aggregated with the galactose binding lectin ricin agglutinin. Under conditions where more than 1% (mole) glycolipid is required to support the aggregation of the smallest liposomes, those of intermediate size require only 0.18% (mole) while the largest liposomes examined require between 0.095 and 0.15% (mole) depending on the method of preparation.