Methodology And Theory Research On Rapid Determination Of Plasma Protein Binding Rate By Fluorescence Quenching Method

Objective:At present, there are some ubiquitous problem in research methods of plasma protein binding rate(PBR), such as they required a longer time,only used few drug concentration gradient to analyse, failed to make enough allowance for the effect of different bonding levels in human serum albumin concentration were given varying doses from patient to patient, etc. In order to solve these disadvantages, this article established the methods of Fluorescence quenching method, as well as to obtain further investigation on this topic of PBR.Methods:(1)To establish theory method of analysising PBR by fast fluorescence quenching. The drug-binding mode, the binding constants, the number of binding sites and thermodynamic parameters between drug and BSA were studied by Stern-Volmer equation, Lineweawer-Burk and Van’t Hoff equation. In addition, the equation relation of PBR tied with drug and BSA concentration, the constants and number sites of binding were analyzed. Based on the experiment data, the change and its law of drugs and BSA concentration difference are analyzed.(2) To investigate the influence factors of BSA fluorescence. Its spectra is tested under different conditions of excitation wavelength, methanel volume,acidity, ionic strength,BSA concentration and stability.(3) Fluorescence titration experiment. At different temperature a series of tubes containing various amount of drugs (the same volume and different concentrations) and a definite amount of BSA(same concentration) was measured under the selected experiment conditions, in which BSA as a fluorescence reagent, and Chinese native medicine as fluorescence quenching reagent.Constant temperature keep1h.(4) To measure the interaction by fast fluorescence quenching. Fluorescence spectra were recorded in the wavelength range of300-500nm, the emission slits were set at BSA at278nm or280nm. Based on relevant theory of fluorescence quenching,the interaction of Chinese native medicine with BSA was detailed analysed. In addition, based on the established equation calculated PBR.Results:(1)The change and its law of Chinese native medicine and BSA concentration difference are found. BSA and drugs concentration is P and D, D/P=X PBR=Y, the number sites and constants of binding is n and K. The law is as follow: This formula is visible:D((P,[D]â†'0;and Kâ†'∞,Xâ†'0,so Yâ†'1D》P,[D]â†'∞;and Kâ†'0,Xâ†'∞,so Yâ†'0when the number of binding sites n=1,(2)The influence factors of the binding reaction results show that the fluorescence measurements were carried out in Tris-HCl buffer of pH7.3containing sodium chloride0.1by excitation wavelength278-280nm, BSA(1.0X10-5mol·L-1)fluorescence relatively stable and sensitive, as well as the BSA spectrum is basic stable within2hours. At the same time, the experiment process should be strictly control the volume of methanol keeping1%in the total volume.(3) The interaction of FRAs and two Chinese native medicine with BSA by fast fluorescence quenching show that there are relatively strong binding affinity for the four anthraquinones with BSA, as well as to be high PBR of4FRAs(>90%).Under the experimental concentrations, The ability of4FRAs prevealed to be Rhein> Emodin> Aloe-emodin> Chrysophanol. PBR is in the dynamic change along with the4FRAs and BSA concentration difference. Fluorescence quenching method was suitable for calculating PBR of Putarin. The experiment demonstrates that the quenching mechanism of them is a static quenching process of a complex fomred. Besides, Putarin might has effect on the serum protein micro-circumstance and conformation. Its binding constant was higher and the reaction temperature was advantageous for the binding reaction in a way. The themrodynamic parameters showed that the interaction of them was driven mainly by hydrophobic force. PBR of Putarin is low and about11.1%-44.4%.Low PBR show that the elimination and distribution rate of Putarin was high and difficult of accumulating, while the interactions of the Ginsenoside Rgl with BSA were weak, the binding constants were low and residence time in vivo was short.Conclusion:(1)In the present work, using the methods of Fluorescence quenching method to study the interaction of Chinese native medicine with BSA, obtain the PBR and predict the PBR law of Chinese native medicine and BSA concentration difference with good accuracy. PBR is in the dynamic change along with the ratio of the4FRAs concentration and BSA concentration difference. The PBR tied with drug variety and dosage and protein quantity, drug variety control category, drug dosage and plasma quantity control protein binding rate decreases. It is with the drug concentration increases or it increases with the increase of BSA concentration.(2)Fluorescence quenching method has many advantages, such as simple to handle, quick detection, improved selectivity, accurate, high sensitivity and detected a large sample of white quantity is little. It play an important role in the interaction of Chinese native medicine with BSA and provide important information for researching PBR. It must will be the high-end technology for researching PBR.