Studies on Interaction of Drugs with Protein and Its Appliction
|Keywords||camptothecines sulfonamides porphyrins transferrin serum albumin fluorescence spectrophotometry ~1H NMR spectroscopy FTIR spectroscopy resonance light scattering|
Proteins are basic ingredients in organism, they undertake many physiological functions. Every kind of pharmaceutical needs the proteins to convey. So it is very important to study the interactions between pharmaceuticals and proteins. The content of proteins is one of the most significantly vital signs, and the work for the determination of the proteins has realistic sense. This project is the forward position and hot point in biochemical and biophysical researches, especially to develop high sensitive and low detection limit methods for protein determination.To research the interaction mechanisms between pharmaceuticals and protein, we used several methods included fluorescence, UV-Vis spectrophotometry,1H nuclear magnetic resonance spectroscopy, fourier transform infrared spectroscopy and some others. In my thesis, twelve pharmaceuticals were studied, two kinds of them were also studied as probes of protein for determination and the results were satisfactory. This paper was written in four sections.In the first section, we summarized the structure and character of protein, the research methods for studying the mechanisms and determination of the protein, and the principle of fluorescence.In the second section, the interaction between transferrin and five kinds of camptothecines was investigated by fluorescence and UV-Vis absorption and 1H NMR spectroscopy at three temperatures. The camptothecines were Topotecan hydrochloride (IH), Irinotecan hydrochloride (IH),7-Ethylcamptothecin(ECPT),10-Hydroxycamptothecin(HCPT),7-Ethyl-10-Hydroxycamptot hecin(EHCPT). Strong fluorescence quenching was observed and quenching mechanism was considered as static quenching according to the stern-volmer eqution. The equilibrium constants K0(L·mol-1) at 25℃were K0(TH)=5.78×105,K0(IH)=4.82×104,K0(ECPT)=9.37×103,K0(HCPT)= 4.01×106, K0(EHCPI)=1.027×103.The binding constants KB(L·mol-1) were KB(TH)=1.66×104, KB(IH)=7.35×104,KB(HCPT)=4.22×104, KB(EHCPT)=1.72×104,KB(EHCPT)=3.34×104. According to the Forster non-radiation energy transfer theory, the average binding distance r(nm) between donor (Tf) and acceptor (camptothecinces) were obtained, r(TH)=3.77nm, r(IH)=3.78nm, r(ECPT)=3.62nm, r(HCPT)=3.92nm, r(EHCPT)=3.42nm. According to the thermodynamic parameters, the main sorts of binding force were determined, and the results indicated that the force between Tf and TH belonged to hydrogen bonding or Van der Waals force, the force between Tf and ECPT, IH, EHCPT belonged to hydrophobic interaction, the force between Tf and HCPT was electrostatic interaction. The results of 1H NMR spectrum show that the active groups of EHCPT interacting with Tf are 10-OH and 20-OH, and 10-OH is more active than 20-OH.In the third section, four kinds of sulfonamides and trimethoprim(Trimethoprim) were studied, the mechanism between BSA and them were discussed. The sulfonamides we studied were sulfadiazine(SD),sulfamethyldiazine(SMl),sulfamethazine(SM2), sulfamethoxazole(SMZ).Strong fluorescence quenching were observed and quenching mechanism were considered as static quenching. According to the stern-volmer eqution., the equilibrium constants K0(Lmol-1) and the binding constant n were obtained,for example the constant at 25℃were listed:K0(SM2)=2.31×105, K0(SM1)=6.92×104,K0(SMZ)=8.16×103,K0(TMP)=4.92×102,K0(SD)=3.32×105. n(SM2)=1.1758, n(SMl)=1.0332, n(SMZ)=1.0331, n(TMP)= 0.7461, n(SD)=1.2457. The average binding distance were r(SM2)=1.85nm, r(SM1)=2.80nm, r(SMZ)=2.59nm, r(TMP)=2.67nm, r(SD)=2.99nm. According to the thermodynamic parameters,the main sorts of binding force were determined, and the results indicated that the force between BSA and SM1,SM2,SMZ,SD were all belonged to hydrogen bonding or Van der Waals force. And the force between BSA and TMP was electrostatic attraction. The investigation of the FTIR spectra of the BSA and SM1,SM2 showed that the conformation of BSA was changed by SM1 or SM2.In the fourth section two kinds of resonance light scattering(RLS) probes of protein were studied, the RLS intensity was enhanced by them and the enhanced intensity was in proporition to the concentration of protein. Based this property, a new method for determination the protein was founded and be evaluated.These two probes were sodium-copper chlorophyllin (SCC) and Methyl pheophorbide-αcopper (MPa-Cu). The method was optimized by changing the factors and under these factors the RLS of protein and SCC or MPa-Cu were studied. With MPa-Cu being the probe, the detection limit was 0.067mg/L, the recovery was 95.6%-102.3%. And the concentration of the HSA in the blood was 64.82g/L, RSD was 1.78%. The result was statisfactory contrast with Coomassie brilliant blue method and Flow Injection-Inhibited Chemiluminescence method. Also with the CCS, the detection limit was 0.0104g/L, the concentration was 61.09g/L, RSD was 1.6%, the recovery was 99.14%-103.12%.