Probing hemoglobin glyco-products by fluorescence spectroscopy

Abstract

Maillard reaction products (MRPs) participate in reactions of carbohydrate intermediates with proteins, resulting in the formation of advanced glycation end-products (AGEs). Dietary Maillard reaction products are recognized as potential chemical modifiers of human proteins. We have investigated the reaction of isolated MRPs from an asparagine-glucose model system with hemoglobin (Hb) to elucidate the binding effect of the MRPs in hemoglobin by fluorescence spectrophotometry. The tryptophan-specific fluorescence obtained for glycated hemoglobin exhibited a Stokes effect since the wavelength of the emission peak was shifted to a higher wavelength than that of native Hb. The formation of new fluorescence emission features indicates the formation of modified hemoglobin species. Fluorescence spectroscopic studies provide evidence that the conformational changes in the β-Trp 37 moiety induce motion of the distal His 64 (E7) in the heme binding pocket. This results in the formation of inactive hemichrome forms of hemoglobin which are related to blood disorders.

Fig. 1. High performance liquid chromatography (HPLC) chromatograms of the reaction mixture of asparagine and glucose (pH 8.0, 180 °C).

Fig. 2. Fluorescence excitation–emission matrix (EEM) of native hemoglobin (pH 8.0) (excitation wavelength range: 260–310 nm).

Fig. 3. Fluorescence emission spectra of hemoglobin with: Panel A: LC Fraction 1, Panel B: LC Fraction 2, Panel C: LC Fraction 3, Panel D: LC Fraction 4 from the reaction of Asn–Gluc after 1 day incubation (excitation wavelength range: 260–310 nm).

Fig. 4. Fluorescence emission spectra of hemoglobin with: PanelA: LC Fraction 1, Panel B: LC Fraction 2, Panel C: LC Fraction 3, Panel D: LC Fraction 4 from the reaction of Asn–Gluc after 1 month incubation (excitation wavelength range: 260–310 nm).

Fig. 5. Fluorescence mapping of hemoglobin with: Panel A: LC Fraction 1, Panel B: LC Fraction 2, Panel C: LC Fraction 3, Panel D: LC Fraction 4 from the reaction of Asn–Gluc after 1 month incubation (excitation wavelength range: 260–310 nm).