Study On Interaction Between Small Molecules And Serum Albumin Using Mass Spectrometry And Spectroscopy

Proteins are the performer of life activities.They can bind many types of ligands,such as fatty acids,metal ions,drugs and surfactants.Among them,surfactants and drugs are widely used in our daily lives.The protein-surfactant complex formed between proteins and surfactants not only has important applications in the fields of cosmetics research and food safety,but it can be also used to simulate biological systems.And under physiological conditions,studying the interaction between proteins and drugs will help us understand the mechanism of drug binding,absorption,metabolism in the human body.Therefore,this thesis individually investigated the interactions of docusate sodium(DSS,a multi-functional anionic surfactant)and baicalin(BA,an anti-inflammatory drug)with bovine serum albumin(BSA)and human serum albumin(HSA)by using electrospray mass spectrometry(ESI-MS),fluorescence spectroscopy(FS),ultraviolet-visible absorption spectroscopy(UV-Vis),circular dichroism(CD)and dynamic light scattering(DLS).The research work of this paper can be divided into three parts.Part Ⅰ:This part uses ESI-MS combined with spectrometry to explain the mechanism of interaction between DSS and BSA.The results of ESI-MS show that a stable protein complex with a stoichiometric ratio of 1:3 can be formed between DSS and BSA.The results of fluorescence spectrum experiments indicate that DSS can strongly quench the endogenous fluorescence of BSA and cause the maximum emission wavelength of BSA to be blue-shifted from 339.2 nm to 329.4 nm,which proves that there is an interaction between the two.Through temperature gradient experiments,it was further determined that the type of DSS quenching fluorescence of BSA was static quenching mode,and the binding constant between the two was calculated to be in the order of 104 L/mol.The van’t Hoff equation was used to calculate thermodynamic parameters of the reaction between the two,and it is speculated that DSS can be incorporated into the hydrophobic cavity in the BSA subdomain IIA through hydrophobic and electrostatic interaction.UV-Vis absorption spectra further proved the existence of static quenching between DSS and BSA.The results of CD and DLS experiments suggest that the presence of DSS can change the microenvironment of Trp residues in BSA,resulting in enhanced hydrophobicity around BSA,which leads to an increase in the α-helix content and a decrease in hydrodynamic diameter in BSA.Part Ⅱ:This part applies ESI-MS combined with multiple spectroscopic methods to study the mechanism of interaction between DSS and HSA in detail.ESI-MS experimental results show that four DSS molecules can be bound to each HS A molecule.The results of fluorescence spectrum indicate that DSS can significantly enhance the endogenous fluorescence of HSA,and at the same time,it can cause the maximum emission wavelength of HSA to be blue-shifted from 334.4 nm to 313.2 nm,which proves that the addition of DSS can alter the microenvironment of Trp residues in HSA.The binding constant between DSS and HSA(greater than 105 L/mol)was calculated by the double reciprocal equation,indicating that there is a strong affinity between the two.The van’t Hoff equation was used to calculate the thermodynamic parameters between the two.Furthermore,according to Ross’s law,DSS can be combined with the"Sudlow’s Site I" in subdomain IIA of HSA by hydrophobic and electrostatic interaction.The results of CD and DLS further suggest that the hydrophobic chain of DSS can provide a more hydrophobic environment for HSA,resulting in an enhanced hydrophobic interaction between the two,which eventually leads to an increase in the spiral content and a decrease in hydrodynamic diameter in HSA.Part Ⅲ:This part applies ESI-MS and multiple spectroscopic methods to study the interaction between BA and HSA.ESI-MS results show that stable protein complexes with a stoichiometric ratio of 1:2 can be formed between BA and HSA.The results of fluorescence spectrum show that BA can cause significant endogenous fluorescence quenching of HSA,accompanied with a red shift of the maximum emission wavelength of HSA,which proves that BA can reduce the hydrophobicity around Trp in HSA and cause the structure of protein becoming loose.Furthermore,at different temperatures,Stern-Volmer and Lineweaver-Burk equations were used to determine the type of BA quenching fluorescence of HSA.It was found that HSA-BA protein complex was formed between BA and HSA through complexation reaction.The thermodynamic parameters between the reaction of BA and HSA were calculated by the van’t Hoff equation,and it was speculated that BA combined with HSA was mainly through electrostatic interaction.The results of CD and UV-Vis absorption spectroscopy indicate that BA can disturb the secondary structure of HSA,causing the decrease of αhelix content in HSA.DLS results suggest that interaction of BA with HSA will enlarge the hydrodynamic diameter of HSA.