| Hemoglobin (Hb) is a kind of protein that exists extensively in erythrocytes. It contains two a chains and two β chains, and it is a tetramer. Each subunit has a peptide chain and a heme. Hb mainly works as oxygen and carbon dioxide transporter as well as the regulator of blood pH in mammals [1]. Besides, it can interact with many endogenous and exogenous substances. Antibiotics, antivirus, antioxidant and so on are the common bioactivities of flavones and thus it makes them important active ingredients worth to be concerned. Flavonoids are poorly absorbed and rapidly participate in phase II reaction(binding reaction), transforming into polar and hydrophilic conjugates. So that circulating levels of unmodified compounds in vivo are generally low[2]. Some reports[3,4] say that quercetin and resveratrol can partition into red blood cells and bind with membrane and hemoglobin. Cell-associated compounds are biological active[5] and can at least protect from conjugative enzymes and thus demonstrate an extended life time, which will replace the moles eliminated via metabolism and excretion. We studied the interaction between Hb and flavonoids in the article hoping to throw light on the drug effect and work mechanism study in molecular level, and what is the most important should be that it is an essential supplement of pharmacokinetic study.Objective:To study the spectra and the change after flavonoids interacting with Hb, discuss the quenching mechanism, binding constant and the number of binding sites, the non-radioactive energy transfer distance, related thermodynamic parameters, the spectra information that when the conformation of Hb changes, and to investigate the influence of Fe2+and Vitamin C on the interaction.Methods:Under the condition that pH is similar to that of human body, we used Ultraviolet/Visible absorption spectra and fluorescence emission spectra together to investigate the interaction between eight flavonoids (Scutellarin, Baicalin, Luteolin, Hyperoside, Galuteolin, Apigenin, Acacetin, Acacipetalin) and bovine hemoglobin (BHb) under298K and31OK, respectively. We discussed the quenching mechanism and calculated the binding constant and number of binging sites. By overlapping the two spectra and the integral area, we obtained the non-radioactive energy transfer distance. By the thermodynamic equations we got the style of binding forces. We used synchronous and3D spectra to explore the conformation change of BHb. At298K, we investigated the binding constant and conformation change.Results:Baicalin, Luteolin, Galuteolin, Apigenin, Acacetin quenched the fluorescence by dynamic quenching mechanism, and Scutellarin, Hyperoside, Acacipetalin by static quenching mechanism. The order of binding constant from big to small was as:Scutellarin, Baicalin, Luteolin, Hyperoside, Galuteolin, Apigenin, Acacetin, Acacipetalin. The values (L-mol-1) were:1.28x107,4.17x106,2.38x105,2.11x105,1.87x105,1.81x105,7.01x103,1.86x103. The number of binding sites were approximately1. In the process of eight flavonoids interacting with BHb, non-radioactive energy transfer occurred. The interaction between them was spontaneously. The forces between Baicalin, Luteolin, Galuteolin, Apigenin, Acacetin and BHb were mainly hydrophobic interaction. Scutellarin and Hyperoside interacted by electrostatic forces, and Acacipetalin by hydrogen and Van der Waals forces. Synchronous and3D spectra demonstrated that eight flavonoids interacted with BHb, and thus the spectra had red shift and the conformation of BHb changed. By the influence of Fe2+and Vitamin C, there is change of the interaction and the conformation of BHb.Conclusion:Under the physiological pH condition, eight flavonoids interacted with BHb at298K and31OK, respectively. Thus the form of the drug changed into binding form and the drug had a new distribution in the body. The original conformation of BHb changed. In the presence of Fe2+and Vitamin C, there is difference of the binding. |
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