Biological Analysis

Methods for the determination of plasma protein binding

Plasma protein binding rate (binding rate of plasma protein, BRPP) refers to the drug absorption into the blood, most of the binding to plasma proteins, the percentage of therapeutic doses of drugs bound to plasma proteins.

Under normal circumstances, various drugs are bound to plasma proteins at a certain ratio, and both bound and free forms are often present in plasma. And only free drugs have pharmacological activity. When the two drugs are used in combination, the drug molecule with stronger protein binding ability occupies the binding site, so that other drugs cannot be fully combined, so that the free part of the latter increases and the efficacy is enhanced. This kind of interaction is meaningful for some drugs with high protein binding rate. Therefore, we should pay attention to those drugs with strong efficacy or toxicity, so as to prevent the drug from being displaced from the binding site and enhance the efficacy, which is often dangerous. sex.

Plasma protein binding characteristics

The plasma protein binding rate is reversible and loose binding, the molecular weight of the bound drugs increases, and they cannot be transported, metabolized and excreted across the membrane, and temporarily lose their pharmacological activity. The characteristics of the binding of drug molecules to plasma proteins (similar to the binding of drugs to receptor proteins): saturation and reversibility, inactive conjugates, and competitive displacement.

Plasma protein binding rate determination method

Equilibrium Dialysis

Equilibrium dialysis is a traditional and mature membrane separation technique to quantitatively study the binding equilibrium between proteins and small molecules. The working principle of the equilibrium dialysis method is based on the difference in molecular size or weight. The macromolecular solution and the small molecule solution are placed on both sides of a semipermeable membrane that only allows small molecules to pass through and does not allow macromolecules to pass through. Under driving conditions, drug molecules diffuse through the semipermeable membrane. When the dialysis reaches equilibrium, the concentration of small molecules in the solution on both sides of the membrane is measured, and the data of the binding of macromolecules to small molecules can be analyzed by calculation. The method is completed in solution, and the pH and ionic strength of plasma and buffer remain relatively constant, which can reflect the in vivo situation to the greatest extent, and the results are reliable, so it is often used as a classic reference method. Long Youqi et al. used equilibrium dialysis to determine the plasma protein binding rate of propofol microemulsion injection at different concentrations. The results show that propofol microemulsion injection is a high protein binding drug, and its effect on the protein binding rate when the drug is used in combination should be fully investigated to ensure the safety of human medication.
Equilibrium dialysis has the disadvantage of taking too long, usually 12-48 hours. Semipermeable membranes and proteins are prone to volume migration effects, which may produce Gibbs-Donna effects and non-specific drug adsorption effects on the surface of dialysis equipment for charged proteins. At present, a commercial 96-well equilibrium dialysis device is used for the determination of plasma protein binding rate. The device can reduce the non-specific drug adsorption on the surface of the dialysis device, and can accelerate the dissolution of the sample to be tested, shorten the equilibration time, and improve the throughput of sample analysis. . In addition, equilibrium dialysis combined with electrochemiluminescence, fluorescence, and mass spectrometry and other highly sensitive detection methods have been applied to the determination of plasma protein binding. Zhou Lingling et al. used the traditional equilibrium dialysis method to successfully measure the binding rate of disopyramide to human plasma proteins by capillary electrophoresis-electrochemiluminescence technology.


Microdialysis technology is a biochemical sampling and analysis technology of extracellular fluid in vivo. Because of its unique minimally invasive and continuous sampling, it has been widely used in exploratory experiments of various pathophysiological phenomena in brain tissue, neurobiochemical detection and drug metabolism research. The microdialysis method is used for the determination of the plasma protein binding rate of the drug. It is based on the fact that the drug cannot penetrate the semipermeable membrane of the microdialysis with a certain relative molecular weight cut-off value after binding to the macromolecular protein, and the free drug can pass through the semipermeable membrane, so as to indirectly determine the Plasma protein binding of the drug. Since most of the collected are small molecules that cannot penetrate the dialysis membrane, namely free drugs, real-time protein binding rate research can be carried out. The drug loss caused by microdialysis sampling is negligible, which makes the determination result more accurate; the very small sampling volume and sampling volume of the drug ensure that the concentration is always constant during the sampling process, which is its superiority compared with other protein binding rate determination methods. Zhang Yingfeng et al. used microdialysis technology to sample, used HPLC-UV detection, and used zero net flux method to determine the in vitro plasma protein binding rate of sinomenine hydrochloride at high, medium and low concentrations. However, this method can only measure the free concentration of the drug, and cannot obtain information on the total concentration or the concentration of the combined drug.
In recent years, the combination of microdialysis with analytical instruments such as high performance liquid chromatography, capillary electrophoresis or mass spectrometry has shown good development prospects in the study of dynamic changes in life processes. Dong Wenbin et al. used microdialysis combined with high performance liquid chromatography to determine the binding rate of minocycline to rat plasma protein, and obtained the pharmacokinetic parameters of minocycline in rat blood and brain.


Ultrafiltration is similar to equilibrium dialysis, and it is also a common method to study the binding effect of proteins and small molecules. In this method, drug molecules diffuse through a semipermeable membrane driven by a pressure difference. The biggest advantage of ultrafiltration is that it can achieve rapid separation of free small molecules in plasma, and it takes a short time. Compared with equilibrium dialysis, the separation rate is greatly improved. However, this method also has disadvantages such as Gibbs-Donna effect, non-specific drug adsorption effect on the surface of dialysis equipment, and protein leakage. Due to the simplicity of operation, rapid analysis, and the application of a commercial 96-well equilibrium dialysis device, ultrafiltration can be used for drug screening, efficacy tracking, and free drug concentration analysis and drug protein analysis of large-scale biological samples in clinical pharmacology and pharmacodynamic studies. Determination of binding rate.
In addition, Chen Ying et al. used ultrafiltration method combined with high performance liquid chromatography (HPLC) to determine the protein binding rate of oridonin A in rat, rabbit and healthy human plasma. The results show that this method is simple, rapid, sensitive and specific, and can meet the requirements of testing biological samples.


The basic principle of ultracentrifugation is that under the action of a certain centrifugal force field, small molecules and proteins stay in different positions in the liquid medium according to the different sedimentation speeds or densities in the liquid medium. In the determination of the drug protein binding rate, the protein-bound drug forms a precipitate, and the free small drug molecules are quantitatively determined in the supernatant of the centrifuge tube. The advantage of this method is to overcome the shortcomings related to dialysis membranes such as Gibbs-Donna effect and membrane adsorption effect. Li Qiuhong et al. used ultracentrifugation to remove the plasma protein, and used high-performance liquid chromatography to determine the blood concentration of syringoside after high, medium and low dose administration. The results showed that the plasma protein binding rate of syringoside had no mass concentration dependence, and the clinical application of syringamarin had good safety.
Compared with the equilibrium dialysis method, the instrument used in this method is more expensive, and physical phenomena such as sedimentation, reverse diffusion and viscosity affect the accurate quantification of free drug concentration, thus limiting the application of this method.

Spectroscopy-based methods

Spectroscopic techniques are also mostly used for the determination of drug plasma protein binding constants. After the drug is combined with the protein to form a supramolecular complex, the spectral and electrochemical properties of the system are changed, thereby providing information on protein concentration or structure. Commonly used spectroscopic methods include UV-visible absorption spectroscopy (UV-visible), fluorescence spectroscopy (fluorescence), infrared spectroscopy (infrared), metadichroism (circulardichroism, CD), optical rotation (opticalrotatory dispersion, ORD) and nuclear magnetic resonance. Resonance method (NMR). In addition to determining the binding rate of drugs and proteins, these methods can also obtain information on the number of protein and drug binding sites, binding sites, force types, and changes in protein structure and function under the action of drugs. Spectroscopic techniques are not suitable for studying multi-equilibrium systems.
Fluorescence spectroscopy has the advantages of high sensitivity, strong selectivity, and small sample volume. Therefore, fluorescence spectroscopy plays an important role in the study of the interaction between small molecules and proteins. However, this method requires a lot of calculations, which is not simple and intuitive compared with other methods.
Surface plasmon resonance technology is a new technology that is very popular in recent years, and it also adopts a method based on spectroscopic technology. Its basic principle is that when the incident light of a certain angle enters the glass prism of the induction sheet, the generated total internal reflection evanescent wave will resonate with the surface plasmon, and the incident light will be absorbed, resulting in a sharp drop in the reflected light energy. Resonant peaks (ie, the lowest values ​​of reflection intensity) appear on the reflection spectrum. Any slight change in the refractive index of the surface medium will cause a shift in the angle of incidence, which is captured by the detector and translated into the corresponding spectrum. The use of surface plasmon resonance technology to study the interaction of drugs and proteins has the advantages of no labeling and rapid detection. However, the instruments used in this method are complex and expensive, making it difficult to miniaturize. So far, this technology has made a lot of achievements in the research of antibody-antigen reaction, the study of simulated cell membrane and drug action mechanism, the analysis of DNA and protein interaction, and the research of virus detection. is also getting more and more attention.

high performance affinity chromatography

High-performance affinity chromatography is used to determine the binding of drugs to plasma proteins, and the binding constants of drugs to proteins can be calculated from the continuous changes in the rate of change of drug migration. The good precision and reproducibility of the chromatographic system provides comparative studies of a large number of binding interactions and can be easily combined with techniques such as MS. This method allows simultaneous injection of multiple drugs and simultaneous determination of the binding constants of multiple drugs and proteins, which is very useful for the study of stereoselective protein binding.
High-performance affinity liquid chromatography is a powerful tool to study the interaction between small ligands and biological macromolecules, and its stationary phase is an immobilized biopolymer (enzyme, receptor, ion channel or antibody). Affinity capillary zone electrophoresis has the advantages of high separation efficiency, small diffusion coefficient, small sample consumption, simple instrument operation, and the ability to use the same (or similar) system to the living physiological conditions in the study of drug-protein binding.


Microcalorimetry is an important method developed in recent years to study the kinetics of biothermochemical and biochemical processes. Adding any reagents will not interfere with the normal activities and metabolism of the biological system, and has been used in the interaction of some antitumor drugs, biological dyes, and small drug molecules with DNA or proteins.

Research Trend of Plasma Protein Binding Rate

In recent years, with the in-depth study of pharmacokinetics and the development of the bioanalysis industry, the binding rate of drugs to plasma proteins has become a research hotspot in the field of pharmacy, especially for those drugs with high plasma protein binding rates. Although the equilibrium dialysis method is complicated and time-consuming, the analysis cost is low, and the concentration of free small molecules can be directly obtained. It is still a conventional method for determination. Spectroscopy can determine the binding rate of drugs and proteins, in addition to the number of binding sites of proteins and drugs, binding positions, types of forces and information on changes in protein structure and function under the action of drugs, but spectroscopic techniques are not suitable for studying multiple equilibria system. Chromatography is the fastest-growing method to study the binding rate of drugs to plasma proteins in recent years. In particular, capillary electrophoresis has the advantages of simple operation, high separation efficiency, less sample consumption, and research can be carried out in a near-physiological environment. Effective methods of drug-protein binding are being widely studied and applied. However, the sensitivity of conventional UV detectors in capillary electrophoresis is poor, which limits the application research of low drug concentration and protein binding. Therefore, the development of high-sensitivity detectors is still a hot spot in the field of capillary electrophoresis.