The Study On The Interaction Of Biomacromolecule With Small Molecules

Protein as the essential biological material in the cells has been playing many vital roles for all kinds of biological phenomena.It has many important functions such as transportation and distribution,whose biological function is dependent on its special structure.Serum albumin is the most abundant and the most important carrier protein in plasma.To begin with,drugs first interact with serum albumin,and then are transported to different parts of the body to takes effect. Studying the interaction between proteins and small molecule drugs from the level of molecules contributes to understanding the metabolism and transfer of drugs and to provide valuable information of drugs design and development.Exploring the interaction mechanisms on these bio-macromolecules with small molecules or ions,especially for those targeting-drug molecules,at the molecular level is of current interest in such as biology,clinical medicine,medicinal chemistry,chemistry and etc.The dissertation is a part of"The Study on the Interaction of between Serum Albumin,DNA and Anticancer Drug",a project which is supported by Zhejiang Natural Science Foundation(No.202056),and is also a part of"The Study on Thermodynamics Properties under Simulative Physiological Conditions",a project which is supported by National Natural Science Foundation(No.20273061).The present work consists of the following 6 parts.1.In this dissertation,the fluorescence spectroscopy including synchronous fluorescence, three-dimensional fluorescence and the three-dimensional polarization fluorescence combined with U-visible absorption spectroscopy,resonance Rayleigh scattering spectroscopy,Capillary Electrophoresis was used to investigate the interaction of serum albumin with several Quinolones durgs.The following major works were carried out.2.The binding of Lomefloxacin Hydrochloride(LMFX) to Human Serum Albumin(HSA) was investigated by spectroscopic(fluorescence spectroscopy,synchronous fluorescence and ultraviolet spectrum) techniques under pH=7.36 in Tris-HC1 buffer solution.Quenching for intrinsic fluorescence of Human Serum Albumin by LMFX was investigated in different temperatures by fluorescence spectroscopy.Data were handled by using Stern-Volmer,Scatchard Lineweaver-Burk double reciprocal equations and double logarithm equations.The major force between them was obtained by thermodynamic parameters.It was found that non-fluorescence complex formed between LMFX and Human Serum Albumin resulted in the decrease of intrinsic fluorescence of HSA.Thus static quenching predominated in the binding process.The binding constants of the complex formed by LMFX and HSA is 10⁴～10⁵L.mol^-1,which illustrated that the complex is more stable.Thermodynamic parametersΔH,ΔG,ΔS were calculated and their interaction forces were revealed.The distance r between donor(HSA) and acceptor(LMFX) was obtained according to fluorescence resonance energy transfer.Negative entropy(ΔS) and negative free energy(ΔG) indicated that the interaction of HSA and Lomefloxacin Hydrochloride is spontaneous process.Hydrogen bonds and Van der Waals interactions play a major role in stabilizing the complex.The spectral results observed indicated that the binding of LMFX to HSA induced conformational changes in HSA which means polarity around tryptophan residues was increased and the hydrophobicity was decreased.In addition,the binding instance r is 2.85nm, which illustrated there is non-radiation energy transfer between LMFX and HSA.3.The interaction of Fe³⁺ with HSA-LMFX systems has been studied using the UV-spectroscopy fluorescence quenching technology and Resonance Rayleigh scattering spectra. The results show that the binding of Fe³⁺ with HSA-LMFX can result in a significant enhancement of RRS,and the fluorescence quenched of HSA.The fluorescence quenching values (ΔF) and the increments of scattered intensity(ΔI) are directly proportional to the concentrations of Fe³⁺ in a certain range.Resonance Rayleigh scattering(RRS) is a new analytical technique developed in 1990s.It has high sensitivity and good selectivity in studying the aggregation of chromophores on biological macromolecules and the ion-association between the anions and cations,and in studying the nonbonding action of biological macromolecules,in particular. Because of these merits,RRS has been used as a useful method for the study and determination of biological macromolecules,and this new technique has showed great advantages in the researches of nucleic acids.With the development of molecular biology,people pay extensive attentions to studying the modes of interaction of small molecules with biological macromolecules at the molecular level.4.The technology of three-dimensional fluorescence spectrometry is developed and has been used to study the conformation change of HSA after the binding interaction of these quinolone drugs(Lomefloxacin Hydrochloride,Pefloxacin Mesylate,Sparfloxacin,Gatifloxacin) with HSA. The results show that quinolone drugs could be used not only the polarity of microenvironment but also the organization of microenvironment by the fingerprinting characteristics of three-dimensional fluorescence spectrometry.5.The binding distance of fluorescein to bovine serum albumin(BSA) and the hydrodynamic radius of BSA in urea-water mixtures were determined by a fluorescence quenching technique and dynamic light scattering measurements respectively,and utilized to investigate the interaction between urea and the protein and its influence on the conformation of the protein in aqueous solution,together with the analysis of the fluorescence spectra of BSA and fluorescein in the ternary mixtures of BSA-urea-water and fluorescein-urea-water and the quaternary mixtures of BSA-flurescein-urea-water.The results showed that the three domains of BSA were of different stability in urea-water mixtures,i.e.,domainâ…¢was unstable even at a low urea content and domainâ… andâ…¡were unfolded above urea concentration of 3.0 and 4.0 mol·L^-1 respectively. Domainâ…¡of BSA was found to be of more compact conformation in the mixtures up to the subdenaturating concentration of urea,which was attributed to the effect of "protein stiffening" resulting from the binding with urea.6.Densities of glycine,glycylglycine,triglycine in sucrose-water mixed solvent have been measured at 288.15,298.15 and 308.15 K by Anton Paar DMA55 vibrating-tube digital densimeter.The apparent molar volume V_φ,limiting partial molar volume V_φ⁰,partial molar volumes of transferΔtrsV_φ⁰ from water to sucrose-water mixed solvent and the hydration numbers N_h for them have been calculated.The transfer volumes from water to sucrose-water mixed solvent and hydration numbers have been discussed in terms of the cosphere overlap model.The results show that a dominant structural interaction between the charged centers of the model compounds and sucrose gives positive contribution to the transfer volume.The partial molar volumes of transfer of the model compounds are positive and increase with increasing sucrose concentration while the hydration numbers decrease with increasing temperature and sucrose concentration.The limiting partial molar volume increases with increasing temperature,and partial molar volumes of transfer depends less on temperature.