Biointeraction analysis of carbamazepine binding to α₁‐acid glycoprotein by high‐performance affinity chromatography

Abstract

Interactions of the drug carbamazepine with the serum protein α1-acid glycoprotein (AGP) were examined by high-performance affinity chromatography (HPAC). Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low affinity interactions with the support and high affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (± 0.1) × 105 M−1, with values that ranged from 5.1 to 0.58 × 105 M−1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how HPAC can be used to examine biological interactions and drug-protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies

“…(1) can be used to describe the relationship between the apparent moles of the target analyte that are needed to reach the mean point of the breakthrough curve ( m Lapp ) as a given molar concentration of the applied analyte, [A] [1,17,27]. …”

“…According to Eqn. (1), a system with single-site binding would be expected to produce a linear response for a plot of 1/ m Lapp vs. 1/[A], where the values of m L and K a can then be determined from the slope and the intercept of this plot [27,41]. …”

“…For instance, Eqn. (2) relates m Lapp to [A] for a system in which two types of independent binding sites (L 1 and L 2 ) are present for A in the column [27,41]. …”

“…Eqn. (2) can be used to obtain information for a two-site system on the amount of each site that is present (i.e., m L1 and m L2 ) and the association equilibrium constants for these sites ( K a1 and K a2 ) by using a non-linear fit to a plot of m Lapp vs. [A] [27,41]. …”

Monitoring binding affinity between drug and α1-acid glycoprotein in real time by Venturi easy ambient sonic-spray ionization mass spectrometry

“…Over the past few decades, several methods have been applied to analyze drug-protein binding affinity, including affinity chromatography [7,8], solid-phase microextraction [9], ultrafiltration [10], and surface plasmon resonance [11]. Although traditional offline methods are widely used in the study of drug-protein affinity, the use of organic solvents tends to change the three-dimensional structure of proteins.…”