Study Of Interaction Between Serum Albumin And Compounds By Spectrometric Methods

Serum albumin,as the most abundant protein in plasma,plays a dominant role in the transport and deposition of a variety of endogenous and exogenous substances in circulatory system.It is closely related to the transport,distribution and metabolism of compounds in vivo.Therefore,the study on the interaction between serum albumin and compounds provides enhanced support for the pharmacokinetics,pharmacological effects,pharmacodynamics and toxicology of drugs.Human serum albumin(HSA)and bovine serum albumin(BSA)have become most studied model proteins in ligand-protein interaction study.The binding strength and main forces of compounds in the interaction process and the serum albumin binding rate affect their bioactivity.Usually,spectrometric method is used to explore the mechanism of interaction between serum albumin and small molecules because of its high sensitivity,strong selectivity,convenient operation and less reagent.While,the determination of serum albumin binding rate is limited to present method that is time and sample consuming.In addition,the binding rate and the interaction mechanism have been rarely studied at the same time.Based on the significance and the research trend in the interaction between small molecules and protein,the interaction between compounds and serum albumin was studied from the following several aspects:(1)Studies on the interaction of serum albumin with the flavonoids,riociguat,piperquine and its metabolites were investigated by spectroscopic methods,and the structure-activity relationship were analyzed.Those compounds were selected according to the binding capacity.(2)Practical sample pretreatment methods based on centrifugal ultrafiltration were developed to determine serum albumin binding rates by high performance liquid chromatography(HPLC).(3)The conversion formula of serum albumin binding rate and fluorescence quenching constant was derived,and the calculation were compared with the data of traditional methods.Besides,the feasibility of predicting serum albumin binding rate by spectroscopic method was also discussed.Research on the interaction between flavonoids from Woodwardia unigemmata and serum albumin was described.A phytochemical study focusing on the flavonoids of W.unigemmata was carried out,which finally led to the isolation of seven flavonoids compounds(1-7).The structures of all compounds were established mainly by NMR and MS experiments,of which compound 1 was identified as a new compound.The multi-spectroscopic methods were used to study the interaction between serum albumin and flavonoids from W.unigemmata.The results indicated that the flavonoids quenched the intrinsic fluorescence of albumin through static quenching.The hydrophobic interactions played a major role in the interaction between compound 1 and serum albumin.The hydrophobic interactions also played a major role in the interaction between compound 7 and serum albumin.Hydrogen bonding and van der Waals forces were the major forces of interaction between compound 2-6 and serum albumin.Conformation of albumin was changed by compound 1-7,and α-helix content was also increased.The results of site competition experiment and molecule modeling method showed that binding site of compound 6 was site I.The multi-spectroscopic methods were applied to study the mechanism of the interaction of serum albumin with piperaquine and its metabolites.The results indicated that piperaquine and its metabolites quenched the intrinsic fluorescence of serum albumin by static quenching.Hydrogen bonding and van der Waals force were the major forces of the piperaquine and the N-oxidated metabolites(M1 and M2),while the hydrophobic interactions were the main forces of the carboxylic product(M3),N-dealkylated metabolites(M4 and M5).The formation of PQ-serum albumin and metabolites-serum albumin led to the change of protein structure and increase of a-helix content.And the binding sites of piperaquine and its metabolites were site I.The interaction between heme and piperaquine and its metabolites was studied by nuclear magnetic resonance spectra.The results showed that the signal spectra of piperaquine and its metabolites were widened by the heme,suggesting that anti-malarial mechanism of piperaquine and its metabolites were related to the combination with the heme.The multi-spectroscopic methods were used to explore the interaction of serum albumin with riociguat.The results indicated that the mechanism of riociguat quenching intrinsic fluorescence of serum albumin is static quenching.Hydrogen bonding and van der Waals force dominated the formation of riociguat-serum albumin complex.Conformation of albumin was changed and a-helix content was increased by the addition of riociguat.The results of site competition experiment,19F NMR and molecule modeling method showed that binding site of riociguat was site I.The practical sample pretreatment methods based on centrifugal ultrafiltration were developed to determine serum protein binding rates of those compounds by using HPLC.The binding rate of kaempferol 3-O-a-L-(3-O-acetyl)rhamopyranoside-7-O-a-L-rhamnopyranoside(KR)and serum protein was 70%,and the binding rate of piperaquine and serum protein was up to 98%,and its N-oxidated metabolites(M1 and M2)were slightly weaker,and its carboxylic product(M3)and N-dealkylated metabolites(M4 and M5)were obviously weak.The binding rate of riociguat and the serum protein is about 95%.The conversion formula of serum albumin binding rate and fluorescence quenching constant was derived,and the calculation were compared with the data of HPLC.The predicted results of KR(medium binding affinity)were different from the results of HPLC analysis.While the calculations of riociguat,piperaquine and its metabolites coincided with the results of determination by HPLC.Thus,it was concluded that the conversion formula could be used to predict the serum albumin binding rate of compounds with high binding affinity.While the prediction of serum albumin binding rate of compounds with lower binding affinity was inaccurate.So further study is required to modify the conversion formula.