Study On The Interaction Between Proteins And Some Drugs Such As PTB And Flavonoids In Folium Ginkgo

The interaction between drug moleculars and biomoleculars, especiallydrug and serum albumin, has direct influence on the distribution andpharmacodynamics of drugs. So the study on the interaction of drugs andproteins becomes the popular issue on pharmaceutics and analytical chemistry.In this paper, two types of the interaction between drugs and proteins have beenstudied. One is the breaking effect. Another is the binding effect.In the experiment of breaking effect, the new compouned PTB, which canbe used to treat the syndromes of diabetes in the future, was used as theobjective molecular. The deleterious AGEs (advanced-glycation end-products)that can be generated in the diabetics become the new target of treating diabetes.PTB is just what has an effect on this target. The breaking effect of PTB onAGE crosslinks has been studied. Meanwhile the binding effect between PTBand HSA(HSA) has been investigated. In addition, the combination betweenHSA and flavonoids in Folium Ginkgo, such as quercetin, chinovose, kaempferoland rutinon, has also been researched.1. PTB had been synthesized, and it is confirmed to be the targetcompound by using NMR and the determination of the disassembly temperature.After albumin has been incubated with glucose at 37 ℃, it has been certifiedthat fluorescent AGE has been produced in vitro by using fluoroscopy and theprotein crosslinks have been generated by using SDS-PAGE. Then PTB hasbeen added to these solutions. The changes of the concentration of AGE and itscrosslinks have been analyzed by using fluoroscopy and SDS-PAGE . Theconclusions are as follows: PTB has no breaking effect on fluorecent AGEs,and the high concentration of PTB has obvious breaking effect on AGEcrosslinks. This effect is stronger at first 4 hours, but weaker from 4 to 11 hours.The middle concentration of PTB and the lowest one have stronger effect at 0 to4 hours, but little effect at 4 to 11 hours. Evenmore, the concentration of AGEscrosslinks have increased to the primary one. The reason of thesephenomenon may be related to the recombination of the AGEs that has beenbroken up primarily. With high concentration of PTB, the breaking effect isstronger than recombination. While at the low concentration, the breaking effectis weaker than recombination. This experiment is in vitro, the splittingfragments can not be cleared. Because these fragments can be cleared in vivo,the recombination may not happen.2. The binding effect between PTB and BSA(HSA) is studied by usingfluorescopy and ultraviolet spectroscopy. The results revealed that thefluorescence quenching of PTB to HSA(BSA) has linear relation with the drugconcentration. According to the slope of the linearity, the quenching constantshave been deduced. Because all these constants are greater than 2.0×1010, thefluorecence quenching are the static quenchings, rather than the dynamiccollision quenchings. This result has been testified by the decrease of bindingconstants with the increasing temperature. According to the quenchingconstants and the characteristics of static quenching, three method ofcalculation of binding constants have been derived. From the slope and theintercept of the lines, the binding constants and the number of binding siteshave been calculated. Because the errors of the outcomes obtained by method 2are greater, these calculation results are neglected. From the outcomes usingmethod 1 and 3, the binding constants at 37℃ are smaller than that at 15℃,which is coincident with the characteristics of static quenching. In method 1, itis supposed that the concentration of the binding drug is far less than the totalconcentration of the drug. In method 2, all concentration of drug are replaced bythe equivalent concentration, which cause great error,especially the error on thenumber of binding sites. However, there is not any suppose in method 3. So theresults calculated by method 1 and 2 are not as accurate as the ones calculatedby methode 3. By using method 3, the binding constants between PTB and BSAor HSA at 15℃ are 3.66×103 and 3.83×103, respectively. The numbers ofbinding sites are 1.02 and 1.16. The percentage of binding are 0.37% and 0.44%.At 37℃, the binding constants are 3.58×103 and 3.35×103, respectively. And thenumbers of binding sites are 0.95 and 0.87. The percentage of binding are0.34% and 0.30%.Moreover, their binding forces are all electrostatic forceaccording to thermadynamic constants. Using the F?ster nonradiation energytheory,it is deduced that the distance between the binding site and tryptophanare 7.5nm and 7.9nm respectively. According to the structure of serum albumin,it can be infered that subdomain ⅡA was the binding sites for the interaction ofPTB and serum albumins, which is near the regions of Try214. The binding siteis out of the hydrophobic area of albumin.3. The binding effect between HSA and the main flavonoids in gingkgosuch as quencetin, isorhamnetin, kaempferol and rutin, has been studied. Thevalues of their binding sites, binding constants, binding force and bindingdistance have been calculated. The results revealed that the fluorescencequenchings of these flavonoids to HSA have linear relation with the drugconcentration. Because all these constants are greater than 2.0×1010, thesefluorecence quenchings are the static quenchings. It is accutate to calculatebingding constants and the number of binding sites by method 3, so method 3 isused directly in this experiment. The binding constants of quercetin and HSA at15℃ and 37℃ are 3.27×104 and 3.24×104. And the numbers of binding sites are0.97 and 0.98. The percentage of binding are 3.07% and 3.17% respectively.The binding constants of rutinon and HSA at 15℃ and 37℃ are 4.18×104 and5.02×104. And the numbers of binding sites are 1.11 and 1.03. The percentageof binding are 4.43% and 4.92% respectively. The binding constants ofchinovose and HSA at 12℃ and 37℃ are 6.60×103 and 5.90×103. And thenumbers of binding sites are 0.90 and 0.81. The percentage of binding are0.59% and 0.48% respectively. The binding constants of kaempferol and HSAat 12℃ and 37℃ are 3.24×104 and 2.74×104. And the numbers of binding sitesare0.82 and 0.99. The percentage of binding are 2.59% and 3.57% respectively.Their main binding forces are all electrostatic, except that the main bindingforce between rutin and HSA is hydrophobic. These results play great roles onthe research of drug distribution and pharmacodynamics. And they are alsouseful in drug designing.