Stepwise frontal affinity chromatography model for drug and protein interaction

Abstract

Frontal affinity chromatography is an efficient technique that combines affinity interaction and high-performance liquid chromatography, and frontal analysis has been used in studying the interaction between drugs and proteins. Based on frontal analysis, stepwise frontal analysis has been established. The present study aimed to use the Lineweaver-Burk plot in stepwise frontal analysis by taking the weighted average of time data. Commercial human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP) columns were used as an affinity column. Warfarin and digitoxin were chosen as model drugs for the HSA column, whereas verapamil and tamsulosin were selected as model drugs for the AGP column. The time data obtained by frontal analysis and stepwise frontal analysis were compared, and the results revealed good correlation (r = 0.9946-0.9998). Frontal analysis and stepwise frontal analysis were also used to analyze the equilibrium dissociation constants (K) of model drugs on the HSA and AGP columns. The K values were compared with literature values, which revealed the same order of magnitude. These results illustrate that conversion of the time data is reasonable and feasible. The Lineweaver-Burk plot can be used in the stepwise frontal analysis model to study the characteristics of the interaction between drugs and proteins. Graphical abstract ᅟ.

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

“…Columns containing human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP) have been employed to determine the equilibrium dissociation constant K d for warfarin- and digitoxin-HSA and verapamil- and tamsulosin-AGP interactions by direct FAC and stepwise frontal analysis. K d values obtained through the different approaches correlate well with literature values, evidencing that the modified staircase method can be applied to assess the binding constants (He et al, 2018).…”

“…A frontal chromatography assay called modified staircase method is an alternative strategy to assess K d and B t . The washing and equilibrium steps between the individual analysis of each evaluated concentration in FAC assays is time-consuming, and the modified staircase (or stepwise frontal analysis) (He et al, 2018) constitutes a promising method to determine the binding constants (de Moraes et al, 2016). In this assay, the ligand is sequentially infused until saturation by a series of low-to-high concentrations is achieved, forming a staircase pattern; with simultaneously infusion of a void marker at a fixed concentration.…”

Immobilization of the SARS-CoV-2-receptor binding domain onto methacrylate-based monoliths for nano LC at 30 nL min−1 and application for research of its ligands

“…3A). In this domain of concentration where the non-specific interactions were negligible, the total amount of PGG interacted within each binding site of the RBD protein and the type of interaction could be characterized by plotting a double reciprocal curve 1/ x versus 1/[PGG] 22 (see eqn (2)). The corresponding plot of the reciprocal of the quantity bound to RBD versus the reciprocal of the corresponding concentration (Fig.…”

“…For each ligand concentration [L], the breakthrough allowed the determination of the amount of ligand x bound to the binding sites of the affinity column, i.e. , the amount of ligand needed to reach the midpoint of the curve according to the equation: 22 x = τ × F × [L]…”

“…The number of mole m L of the active binding sites on the affinity column for ligand L and the dissociation equilibrium constant K d between the ligand and its binding site were determined according to the equation: 22 1/ x = K d /( m L × [L]) + 1/ m L …”