HSA binding of HIV protease inhibitors: a high‐performance affinity chromatography study

Abstract

The binding of HIV protease inhibitors, drugs important for anti-HIV chemotherapy, to HSA was examined by high-performance affinity chromatography. Frontal analysis was first used to determine the amount of anchored protein and the binding capacity for selected markers on this column. Zonal elution experiments then ranked the HSA bound fraction of the examined compounds. Information on the binding region was obtained by competitive zonal elution experiments using probe compounds with known sites on HSA. An allosteric competition between HIV protease inhibitors (PIs) and valproate (a probe for the bilirubin site) was detected, consistent with a noncooperative binding mechanism. No significant competition was observed between the examined compounds and salicylate or ibuprofen, probes for sites I and II, respectively. The observations were confirmed by circular dichroism spectroscopy, based on the change in the induced circular dichroism signals of selected markers for the main binding sites of HSA when ritonavir was added as the competitor. These results were in good agreement with previous literature reports and provide more details on how PIs are transported in plasma and how they may compete with other drugs in the body.

Kinetically-controlled mechanism-based isolation of metabolic serine hydrolases in active form from complex proteomes: butyrylcholinesterase as a case study

“…Temperature and pH of the wash buffer did not signicantly affect the yield, but warm buffer and pH 7 removed the contaminating proteins more efficiently than cold buffer and pH 5 ( Table 3). Neither the increase of the wash volume, nor the addition of NaCl (Table 3) to the wash buffer or 41 The lack of effect of HSA ligands, together with the results obtained from the denaturation experiments and the increase of contaminants observed over time in the load step are consistent with the hypothesis that the retention of HSA and Igs could be a The effect is reported in percentage normalized to the yield and apparent purity obtained in the reference condition. a The pre-elution step was performed at 4 C. Hx, hexanoic acid 3 mM; IBU, rac-ibuprofen 3 mM; SAL, salicylic acid 3 mM; 4MAC, tetramethylammonium chloride 500 mM.…”

Reversible human serum albumin binding of lipocrine: A circular dichroism study

“…The stock solution of rac ‐LIPO (200 μM) was prepared in 1‐propanol, diluted with potassium phosphate buffer (pH 7.4, 67 mM) down to10 μM, and then injected into a silica‐based chromatographic column containing HSA covalently immobilized (Pistolozzi et al, manuscript in preparation). The column was prepared and validated according to previously reported procedure 21. The mobile phase was composed of potassium phosphate buffer/1‐propanol (92:8 v/v).…”

“…If this is the situation, the single enantiomers act as distinct compounds, because their metabolism, distribution, and elimination are, in principle, different. Thus, the impact of enantioselectivity on biological activity becomes evident, and this determines an increasing interest to the identification of the molecular mechanisms involved in the enantioselective drug binding to serum proteins 9–13. AGP is also important as serum carrier, because many drugs bind this protein with quite high affinity 14–16…”

Chromatographic analysis of drug interactions in the serum proteome

“…108 The same method has been used with other pharmaceuticals, 109 including anti-HIV chemotherapy drugs, 110 and has been combined with LC/MS for the simultaneous examination of the protein interactions of several drugs in a mixture. 107 Related reports have used k as a direct measure of the global affinity of injected solutes for an immobilized protein.…”