Preliminary Exploration In The Interactions Of Four Methylphenanthrene Isomers With Different Methylated Positions To Human Serum Albumin And The Related Mechanisms

Alkylated polycyclic aromatic hydrocarbons（A-PAHs）are important derivatives of parent polycyclic aromatic hydrocarbons（P-PAHs）in environment,especially in oil contaminated areas.The persistance and toxicity of A-PAHs are generally greater than their P-PAHs.Methylated PAHs（M-PAHs）with 3～5 benzene rings generally make a remarkable contribution to the toxicity of A-PAHs in oil-contaminated environment.The toxic effects of these M-PAHs on aquatic organisms varied dramatically with their methylated positions.The M-PAHs in environment can also enter human body through various pathways and thus may induce diverse toxic effects.However,there have been few relevant reports.Polycyclic aromatic hydrocarbons（PAHs）can bind to human serum albumin（HSA）,the important transport protein in human plasma.The binding of PAHs to HSA can impact the transportation and distribution of PAHs in human body,which further affect their metabolism and toxic behaviours.Moreover,the HSA-PAH binding generally cause conformational distortions and functional changes of HSA,and thus induce toxic effects.Therefore,studies on the molecular interactions of M-PAH isomers methylated at different positions with HSA,as well as the corresponding effects on the conformation and physiological functions of HSA,will be benefit to reveal the differences in transportation,distribution,and potential biological effects of M-PAH isomers in human body.The cheaper bovine serum albumin（BSA）was usually used as an alternative for HSA in the intermolecular interaction studies,due to its low cost and high similarity in structure with HSA.However,it was revealed that the strength of interactions of the two proteins with P-PAHs can be significantly different,because of the difference in tryptophan（TRP）residues and hydrophobicity of the two proteins.Thus,what’s the difference in the strength of interactions of M-PAH isomers methylated at different positions with BSA?What are the difference and similarity between the interactions of M-PAH isomers with BSA and that with HSA?In this study,methylphenanthrenes（MPs）,the typical 3-ring PAHs,were selected as the object compounds,including three isomers（1-MP,4-MP and 9-MP）methylated at a position（α-MP）and one（3-MP）methylated at β position（β-MP）.The main contents and results of this study are shown as follows:（1）Fluorescence,ultraviolet-visible（UV-vis）absorption spectroscopic methods and molecular docking technique were employed to investigate the interactions of MPs with HSA in simulated physiological conditions（pH=7.4）.The quenching constants（KSv）and quenching rate constants（Kq）of HSA fluorescence decreased with the rising temperature;all the Kq values were much larger than 2.0×1010L·mol^-1·s-1;all the dynamic quenching constants KD values were much smaller than the Ksv(KD（max）=1.05×10⁴ L·mol^-1,Ksv（min）=2.22×10⁵ L·mol^-1,298 K).Those results revealed that,the quenching of HSA fluorescence induced by the four MPs was dominated by static mechanism.The formation of 1:1 HSA-MP ground-state complexes was proved by the alteration of HSA UV-vis absorption spectrum induced by MPs（C（HSA）:C（MP）=1:1）.The results of fluorescence quenching measurement indicated that,the binding constant（Kb）values at 308 K were in the order of 3-MP(9.13 ×10⁴ L·mol^-1)>9-MP(3.57×10⁴ L·mol^-1)>4-MP(2.37×10⁴ L·mol^-1)>1-MP(1.46 ×10⁴ L·mol^-1),and the β-MP has a stronger binding affinity than α-MP at different temperatures（298 K,308 K,318 K）.The negative values of Gibbs free energy change（△G）,enthalpy change（△H）and entropy change（△S）indicated that:the HSA-MP binding processes were spontaneous,exothermic and enthalpy-driven,and the main binding force was van der Waals force.The results of binding site competition experiment revealed that,the binding site of 4-MP in HSA molecule was in the hydrophobic microzone where TRP located in subdomain ⅡA,while the other three MPs bound to the site between subdomains ⅠB and ⅡA.The Kb values of HSA-MP complexes obtained through molecular docking were in the order of 3-MP>9-MP>4-MP>1-MP（298 K）,as same as the results of fluorescence quenching studies.The binding free energy of each binding force calculated through molecular docking reconfirmed that the main binding force of each HSA-MP complex was van der Waals force.The binding sites of four MPs in HSA molecule were visually reconfirmed by the HSA-MP docking conformations and were consistent with the binding sites obtained by competitive experiments.（2）The effects of MPs on the α-helix structures and vitamin B2（VB2）-transporting function of HSA were studied through circular dichroism（CD）and fluorescence measurements in simulated physiological conditions.CD spectra revealed that,theα-helix content of HSA was reduced to 28.15%,34.29%and 40.90%from 53.03%due to the addition of 1-MP,4-MP and 9-MP（C（MP）:C（HSA）=8:1）,respectively,while no significant change in the presence of 3-MP（P>0.05）.The results of transportation function experiment indicated that the Kb value(2.67 ×10⁵ L·mol^-1)of HSA-VB2 complex decreased to 4.69 × 10⁴ L·mol^-1 and 5.28 × 10⁴ L·mol^-1 in the presence of 3-MP and 9-MP,respectively;inversely,increased to 2.82 × 10⁵ L·mol^-1 and 4.24×10⁵ L·mol^-1 in the presence of 1-MP and 4-MP,respectively.The effect degrees of MPs on VB2-transporting function of HSA were in the order of 3-MP>9-MP>4-MP>1-MP,exhibiting a positive correlation with the binding affinities of MPs to HSA.（3）A comparative study on the strength of HSA-MP interactions and that of BSA-MP interactions were implemented through fluorescence quenching measurement in simulated physiological conditions.There were differences betweenthe strength of HSA-MP interactions and that of BSA-MP interactions.1)The KSV and Kq values of HSA fluorescence were significantly greater than that of BSA.2)The Kb values of HSA-MP complexes at 298 K were in the order of 3-MP(1.73×10⁵ L·mol^-1)>9-MP(1.01×10⁵ L·mol^-1)>4-MP(6.72×10⁴ L·mol^-1)>1-MP(2.82×10⁴ L·mol^-1),while that of BSA-MP complexes were in the order of 3-MP(1.77×10⁵ L·mol^-1)>4-MP(4.10×10⁴ L·mol^-1)>9-MP(3.02×10⁴ L·mol^-1)>1-MP(8.12 ×103 L·mol^-1),which indicated that the Kb values of HSA-α-MP complexes at 298 K were in the order of 9-MP>4-MP>1-MP,while that of BSA-α-MP complexes were in the order of 4-MP>9-MP>1-MP;moreover,the binding of α-MP to HSA was stronger than that to BSA.In addition,there were similarities between the strength of HSA-MP interactions and that of BSA-MP interactions:the binding interactions of β-MP（3-MP）to both HSA and BSA were stronger than that of α-MP to both HSA and BSA.This thesis preliminarily revealed the interaction mechanisms of MP isomers differing only in methylated positions with HSA,as well as the differences in binding strengths and binding sites of HSA-MP complexes,through spectroscopic and docking studies on molecular interactions of MP isomers with HS A.These results are benefit to explore the potential differences in transportation and distribution of MP isomers in human body.Moreover,the different effects of HSA-MP interactions on the α-helix structure and VB2-transporting function of HSA were revealed by the results of CD spectra and transportation function experiment.The results are favorable for predicting the potential human health effects of MP isomers and the corresponding difference,and are helpful to understand the relationship between conformation and physiological function of HSA.Besides,the difference and similarity between the strength of HSA-MP interactions and that of BSA-MP interactions were revealed by the results of fluorescence quenching experiments,which provide experimental supports in part for further revealing the influence of albumin structure on the interactions of albumins with A-PAHs.