Studies On Interaction Of Serum Albumin And Drug Molecules By Fluorescence Spectrometry

Serum albumin is the most abundant protein in the plasma and a kind of majorcirculatory protein. The surface of the protein has multiple lipophilic binding sites,which may combine with hydrophobic substances, such as drugs, especially lipophiliccompounds with neutral and negative charge. The interaction of protein and drug playsan important role in pharmacokinetics and pharmacodynamics of the drugs. In thebinding of protein and drug, the non-covalent molecular interaction is widely acceptedas the principal binding mode. Its concentration reaches40mg mL-1(0.6mM),accounting for60%of all proteins in plasma total, providing80percent of the bloodosmotic pressure. In addition, due to intrinsic fluorescence, serum albumin can becombined with many endogenous and exogenous compounds play a role in the storageand transport. When the drug molecules enter the organism, it reached the receptor site,and thus following with pharmacological effects through the storage and transport ofplasma.In recent years, research and application of serum albumin interaction betweenmacromolecules and small molecule drug has been the subject of interest. And as animportant issue, medicine, pharmacology, chemistry, molecular biology and lifesciences, it caused universal attention in many scholars. It has significance in the clinical treatment, pharmacodynamics and pharmacokinetics and also important guidingsignificance for drug molecular design, development of new drugs and so on. Therefore,this thesis work was carried out between interactions of protein macromolecules andsmall drug molecule with fluorescence.Fluorescence spectrometry was applied in the research of interaction betweenprotein and drugs. In the research of non-covalent molecular interactions byfluorescence spectrometry, fluorescence quenching is widely applied. When the methodwas applied, the drugs could not absorb the fluorescence and the binding site numberobtained by the fluorescence measurement of protein, which has intrinsic fluorescenceprobe. And with combined means of fluorescence techniques and absorptionspectroscopy techniques, interaction of serum albumin and drug molecules in vitro werestudied. According to calculation of the binding constants, binding sites number, toobtain the extent of binding, binding force and other external factors impact on thecombination.In this thesis, the interactions of baicalin and wogonoside with human serumalbumin (HSA) were studied by fluorescence quenching and ultraviolet-visibleabsorption spectrometry. The binding constants were obtained by fluorescencequenching of drug with serum albumin. The main acting binding forces of baicalin andwogonoside to serum albumins were hydrophobic force according to thethermodynamic parameters. The effects of metal ions, such as Zn2+, Mg2+, Al3+andCu2+on binding constants were also evaluated. With the combination of fluorescencespectroscopy, absorption spectroscopy and other means, action mechanism of drugs andbiological macromolecules in vitro were studied, following with the degree ofintegration, binding force and combined with the influence of external factors on thedegree of integration and so on. It is not only valuable to reveal the pharmacokinetics,clinical therapy, but also have guiding significance for drug design and development ofnew drugs.A new method was developed for determining the binding constant and number of binding site of protein with drug. The magnetic gold nanoparticles were used for fixingprotein. After the binding of protein with drug, the magnetic goldnanoparticles-protein-drug complex was separated from the free drug with appliedmagnetic field. The free drug concentration was obtained directly by fluorescencespectrometry. The binding constant and binding sites were calculated. This method issuitable for studying the interaction between arctiin and bovine serum albumin (BSA).BSA and arctiin binding constants and binding sites were2.09×105L mol-1and16.63,respectively. With the magnetic field，the immobilization of BSA on the magnetic goldnanoparticles made the BSA be separated from the sample solution. As a result, thereare only drugs in the sample solution and the protein does not affect the determinationof the drugs. So the number of binding sites should be reliable. The results confirmedthat the method is suitable to the measurement of the binding constant and the numberof binding sites of molecular interactions.The thiol resin was used for the immobilization of protein. The binding constant andsite number for the non-covalent complex were directly calculated based on theconcentration of free drugs obtained by fluorescence spectrometry. And the bindingparameters were obtained based on Scatchard equation. The present method was appliedto the investigation of the interaction between BSA and liquiritin. The binding constantand site number for BSA and liquiritin were7.28×104L mol-1and21.0. The obtainedresults confirmed that the present method is a good alternative for the determination ofbinding constant and site number for the molecular interaction. BSA was immobilizedon the thiol resin and the effect of protein on determination of the drugs was eliminatedsuccessfully. The BSA and liquiritin were separately determined and there was not theinterference from each other. The matrices of both standard solution and samplesolution were PBS buffer and the matrix effect on the fluorescent intensity was the samewhen different solutions were measured. It is possible to extend this method to otherquantitative studies of non-covalent molecular interactions by varying the detectionconditions.