Relations between high-affinity binding sites of markers for binding regions on human serum albumin

Abstract

Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1: 1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 104-106M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made.

Five recombinant fragments of human serum albumin—tools for the characterization of the warfarin binding site

“…One high-affinity site and one with lower affinity were found for HSA in agreement with previous reports~Sudlow et al ., 1975a;Maes et al, 1982;Kragh-Hansen, 1985;Larsen et al, 1985;Bos et al, 1989;Kosa et al, 1997!. The order of affinity constants for the native molecule and its recom- The affinity of HSA-DOM IB-II to warfarin was found to be about fourfold lower compared to HSA, leading us to the conclusion that this fragment harbors sufficient information for the formation of the high-affinity warfarin binding site of HSA.…”

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

“…To characterize the site in more detail, several attempts to subdivide the site have been made. 8,10,14) In the crystal structures of HSA and rHSA, site I is formed as a pocket in subdomain IIA (Fig. 1A) and involves the lone tryptophan of the protein ( 214 Trp).…”

“…Endogenous Compounds), and because several examples have been found of independent binding of two different compounds to the site. [7][8][9][10] Several lines of evidence suggest that the site also is very flexible. (i) The fact that ligands with very different chemical structures bind to the region with high affinity indicates that the site is adaptable.…”

Human serum albumin binding ibuprofen: A 3D description of the unfolding pathway in urea

“…The site must be large because ligands as big as bilirubin can be bound, and because several examples of independent binding of two different compounds to the site have been found. [8][9][10][11] Sudlow's site II (located in subdomain IIIA) is preferred by aromatic carboxylic acids with a negatively charged acidic group at one end of the molecule away from a hydrophobic center (e.g., nonsteroidal anti-inflammatory drugs). Site II is composed of all six helices of subdomain IIIA and it is topologically similar to site I but it seems to be smaller, or more narrow, than site I, because apparently no large ligands (e.g., bilirubin, hemin, hematin, or other porphyrins) bind to it.…”