Human Serum Albumin Conformational Changes as Induced by Tenoxicam and Modified by Simultaneous Diazepam Binding

Abstract

The binding of tenoxicam to human serum albumin has been shown by affinity chromatography proton titration and equilibrium dialysis to be dependent on the neutral to basic conformational change of the protein. The influence of diazepam on the interaction was also investigated using the same techniques, suggesting that diazepam increases the association of tenoxicam to albumin. Affinity chromatography revealed that the reciprocal effect also occurs. Displacement studies indicated that diazepam causes a significant increase in the affinity of tenoxicam to its main binding site, albumin site I, which is different from the diazepam site (site II). Tenoxicam seemed to cause an allosteric change in the conformation of the protein during its own binding, as did warfarin. The mechanism of this effect was a pH-dependent conformational change of albumin induced by electrostatic forces within the protein. Diazepam induced a distant accommodation of the protein, an effect accompanied by an enhanced inhibition of the release of protons from albumin.

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin.

“…Drugs: Warfarin 1) 3.4ϫ10 5 10) Diazepam 1) 3.8ϫ10 5 48) Azapropazone 120) 2.8ϫ10 5 9) Ketoprofen 133) 2.5ϫ10 6 136) Phenylbutazone 1) 1.5ϫ10 6 10) Chlofibrate 1) 7.6ϫ10 5 137) Acenocoumarol 10) 2.2ϫ10 5 10) Iopanoate 1) 6.7ϫ10 6 131) Salicylate 1) 1.9ϫ10 5 9) Ibuprofen 22,68) 2.7ϫ10 6 21) Indomethacin 1) 1.4ϫ10 6 129) Pirprofen 72) 3.9ϫ10 5 72) Tolbutamide 1) 4.0ϫ10 4 130) S-Naproxen 1) 3.7ϫ10 6 89) Chlorpropamide 1) 3.3ϫ10 5 9) Ethacrynate 1) 1.7ϫ10 6 34) Iophenoxate 1) 7.7ϫ10 7 131) Chlorothiazide 134) 5.5ϫ10 4 134) Iodipamide 10) 9.9ϫ10 6 10) Diclofenac 76) 3.3ϫ10 6 76) Sulfadimethoxine 121) 9.0ϫ10 4 121) S(Ϫ)-Thiamylal 56) 8.7ϫ10 4 56) Sulfathiazole 9) 2.5ϫ10 4 9) S-Etodolac 59) 2.0ϫ10 5 59) Furosemide 122) 1.9ϫ10 5 122) Carprofen 75) Ͼ10 6 75) Tenoxicam 123) 3.7ϫ10 5 123) Suprofen 68) N.D. R-Sulbenicillin 111) 5.2ϫ10 3 111) Benoxaprofen 22) N.D. Valproate 60) 2.8ϫ10 5 60) Cicloprofen 22) N.D. Piretanide 122) 9.5ϫ10 4 122) Azidodeoxythymidine 15) N.D. Spironolactone …”

Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin

“…Sites I and II are generally considered to be two discrete, well-separated sites, because, in most cases, binding to these two sites takes place independently. However, allosteric interactions between ligands that bind to sites I and II have also been reported, examples of which include warfarin-diazepam [9], ceftriaxone—probenecid or diazepam [10], pinoxicam-diazepam [11], tenoxicam-diazepam [12], aspirin-amlodipine [13] and dansyl-L-asparagine (DNSA)-ibuprofen or diazepam [14]. …”

Determination of Protein Binding of Troglitazone Stereoisomers by Fluorometric Titration

“…These phenomena involving increased¯uorescence intensity are thought to be due to allosteric interactions. 20 All four troglitazone stereoisomers strongly displaced dansylsarcosine, however, only 2R, 5R weakly displaced DNSA, and they did not displace ACAA on HSA (Figure 3). Therefore, the stereoisomers of troglitazone are assumed to be mainly bound to the diazepam site on HSA.…”