Drug - A New Method For Determination Of Protein Binding Constant Research

The research of interaction between protein and drug is one of the hot fields in life sciences and chemistry in recent years. There are many methods to study drug-protein interaction. In this thesis, based on the equation describing the relationship between the injected amount of solutes (nb) and the retention factors (k)(direct injected method), a new method is developed to determine association constants between three kinds of drugs, caffeic acid, wogonin, luteolin, with immobilized bovine serum albumin. The obtained results are as follows:1. The adsorptions isotherm of these three kinds of drugs on the immobilized BSA column were determined by frontal analysis. Two kinds of binding isotherm models, one-site model and two-site model, were used to judge the types of binding site of the tested drugs on BSA. The results showed that there was only a single type of binding site on BSA for three kinds of drugs. Their association constants calculated were1.33×104L/mol,1.29×104L/mol and2.33×104L/mol, respectively.2. Zonal elution was used to investigate the binding interaction of three kinds of drugs with BSA. The results proved the conclusion which three kinds of drugs have only a single type of binding site on BSA. Their association constants calculated were1.16×104L/mol,1.33×104L/mol and1.06×104L/mol, being close to the value from frontal analysis.3. Zonal elution were carried out to investigate binding sites of the four drugs on BSA, in which warfarin, L-tryptophan, digitoxin and clomiphene were used as four probe compounds. Caffeic acid, wogonin, luteolin were found to have direct competition with warfarin, indicating that all the three drugs interacted with sudlow site I (the warfarin-azapropazone site) on BSA.4. Using phosphate buffer as mobile phase, and three drugs, caffeic acid, wogonin and luteolin as injected solutes, association constants of three kinds of drugs with BSA were determined with direct injected methods to be12.42×104L/mol,14.19×104L/mol and57.08×104L/mol, respectively, which were one order of magnitude higher than those from zonal elution and frontal analysis, but close to the literature value of7.01×104L/mol,12.8×104L/mol and48.5×104L/mol. The reasons for inconsistent results between different methods were analyzed.