Studies On The Interaction Of Drug With Proteins

Human serum albumin (HSA) is the most abundant carrier protein in blood circulation, which can bind many endogenous and exogenous substances, such as metabolites, drugs and other biologically active compounds present in blood, and realize transport and distribution of them. Therefore, the investigation of the interaction of HSA with drugs contributes to understand disposition, transportation, metabolism and efficacy of drugs.Lysozyme (14.3 kDa) is a small monomeric globular protein as a bacteriolytic agent having an ability to hydrolyze bacterial cell walls. Catalase is a key antioxidant which detoxifies H₂O₂ by reducing this molecule into molecular oxygen (O₂) and water (H₂O). Lys and CAT also can bind many small molecule substances such as drugs, which has effect on metabolism and efficacy of drugs and enzymic activity. Thus, it has been an interesting research field of life sciences, chemistry and clinical medicine. In this dissertation, on the basis of the previous research, the fluorescence spectroscopy combined with UV-visible absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and computational modeling were used to investigate the interaction of drug with several proteins. The following major innovative works were carried out:1. The interaction of nevadensin with several proteins (HSA, Lys and CAT) has been investigated under simulative physiological conditions. The binding properties including the fluorescence quenching mechanisms, binding constants and the number of binding sites were investigated in detail, the binding distances were calculated, and the main interaction force between drugs and proteins was discussed.2. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed.3. The effects of nevadensin on protein secondary structure were investigated with CD techniques.4. The effects of nevadensin on enzymic activity of Lys and CAT were studied.5. The computational modeling method was used to study the drug-HSA interaction and the study results were in consistent with the experimental results.The paper was clearly divided into three sections:Chapter 1: The structure, functions and properties of protein studied were introduced briefly; The methods and actuality about the research of the interaction between small molecular substances and protein were summarized.Chapter 2: The binding of nevadensin with human serum albumin (HSA) has been studied using fluorescence spectroscopy, CD spectroscopy and molecular modeling methods under simulative physiological conditions. The results of spectroscopic measurements suggested that the intrinsic fluorescence of HSA was quenched by nevadensin through static quenching mechanism. The binding constants, numbers of binding site, and mainly intermolecular force induced by drugs binding were obtained from the fluorescence spectroscopy. The effects of nevadensin on HSA secondary structure were investigated by CD spectroscopy. The study of computational modeling indicates that primary binding site of the drug was located within the subdomain IIA of HSA( site I).Chapter 3: The interaction of nevadensin with lysozyme (Lys) and bovine liver catalase(BLC)have been investigated by fluorescence spectroscopy, CD spectroscopy and measurements of enzymic activity. Fluorescence quenching mechanism of Lys and BLC by nevadensin was discussed in detail. The binding constants, numbers of binding site, and the effect of Lys and BLC secondary structure induced by drugs binding were obtained from the fluorescence spectroscopy, CD spectroscopy. The thermodynamic analysis indicated that hydrophobic interaction can play a main role in the binding process. Further, measurements on the enzymatic activity of Lys in the absence and presence of nevadensin indicated that the interaction nevadensin with Lys and BLC led to a reduction in the activity of Lys and BLC.