Immobilized serum albumin: Rapid HPLC probe of stereoselective protein‐binding interactions

Abstract

A human serum albumin-based HPLC chiral stationary phase (HSA-CSP) has been examined as a tool to investigate binding of chiral drugs to HSA and drug-drug protein-binding interactions. Rac-oxazepam hemisuccinate (OXH) was used as a model compound and the chromatographic retention (k') of its enantiomers was determined after addition of displacers to the mobile phase. Compounds known to bind at the same site as OXH and at different sites were tested for their displacing capacities. Competitive binding interactions between the OXH enantiomers and displacers in the mobile phase were reflected by decreases in the k's of (R)- and (S)-OXH. The results indicate that retention on the HSA-CSP accurately reflects binding to native HSA and the technique can determine enantioselective and competitive binding interactions at specific sites on HSA. The HSA-CSP was also able to recognize separate binding areas for (S)- and (R)-OXH.

Drug binding to human serum albumin: Abridged review of results obtained with high‐performance liquid chromatography and circular dichroism

“…33 Although partial and allosteric competitions have been studied with HSA-HPLC, 36,37 a complete displacement was never reported, even when a large excess of competitor was used. 38 For example, while in solution diazepam is completely displaced from the HSA stereospecific site by a twofold amount of ibuprofen, 2,21,38 the same competition apparently yields incomplete displace-ment on the HSA-HPLC, qualitatively similar to that observed for indirect competitions (Fig. 1A).…”

“…The most retained enantiomer, (S)-OXH, is displaced either by (S)-or (R)-ibuprofen, as expected, assuming a direct competition phenomenon at site II. 38 On the contrary, the retention of the less retained enantiomer, (R)-OXH, does not change significantly in the presence of concentrations of the interacting drug, either (R)-or (S)-ibuprofen, up to 25 lM. Thus, the two enantiomers of oxazepam hemisuccinate possess an independent binding on the protein (i.e., bind at different sites) and this constitutes the basis of the observed enantioselectivity.…”

Quantitative Studies of Allosteric Effects by Biointeraction Chromatography: Analysis of Protein Binding for Low-Solubility Drugs

“…A plot of 1/k versus C I is then made at each concentration of S, which results in a linear relationship for systems fitting the model in Figure 2(a) (Note: it is also possible to use a non-linear fit to the data according to eq 1; however, the linearized form shown in eq 2 is more convenient for routine analysis and for differentiating between systems with competitive binding versus non-competitive binding). 25 The best-fit lines for graphs obtained with eq 2 are then used to make a second graph in which the ratios of the intercepts and slopes are plotted versus the total concentration of solubilizing agent, as described in eq 3, (3) where the reciprocal of the intercept is now K IL, and the ratio of the slope and intercept gives K IS . 19,20 It is possible to simplify this approach if independent estimates are available for K AS and K IS .…”

“…1,2 A common example is the presence of allosteric effects during the binding of drugs or other small solutes to the protein human serum albumin (HSA). [3][4][5][6] Most previous studies of allosteric interactions with HSA and other proteins have involved only qualitative observations of increased or decreased binding. Exceptions include work by Stockton et al 7 and Ehlert,8 in which apparent association equilibrium constants were measured for a receptor with one solute in the presence of several fixed concentrations of a second solute.…”

Enantioselective Retention of 4‐Aryl‐1,4‐dihydropyridine Calcium‐Channel Blockers on Human Serum Albumin and α 1 ‐Acid Glycoprotein HPLC Columns: Relationships with Different Scales of Lipophilicity

“…The study of drug-protein interactions can be facilitated by HPLC methods based on stationary phases packed with serum proteins chemically immobilized on a silica-gel core (biochromatography) [3]. This technique yields binding-related retention data that mirror the drug-binding properties of the corresponding free protein.…”