Nature of Ligand Binding

Selected Binding Constants for Endogenous and Exogenous Ligands with Serum Albumin  

The vast majority of ligands in Table V are bound in one or both sites within specialized cavities of subdomains IIA and IIIA. At this time the binding locations of several key compounds, historically used as markers in drug or ligand displacement interactions, have been determined at various resolutions (Table VI). Clearly, from Table VI it can be seen that IIIA appears to possess the primary binding activity for albumin whereas IIA is more specialized. 

As one might predict, there is enantiomorphic selectivity with regard to various chiral ligands. For example, albumins in general have a 100-fold higher affinity for L-tryptophan (King and Spencer, 1970 La-gercrantz et ai, 1981) and stereoselectivity is also known for many pharmaceuticals such as rf-oxazepam hemisuccinate (Muller and Wollert, 1975). 

Much of the chemistry of albumin can be understood from detailed observations of the 2,3,5-triiodobenzoic acid (TIB) complexes with albumin. The crystalline complex of TIB with HSA and ESA has been determined with resolution sufficient to position the molecule within the binding pocket unambiguously and to identify the chemistry of interaction (Fig. 15, see color insert). This ligand has a moderate and equal affinity for IIA and IIIA in both HSA and ESA. Association constants were estimated by Scatchard analysis (Scatchard, 1949) using direct linear plots (Eisenthal and Cornish-Bowden, 1974) to be 2.2 x 10 M and 8.3 X lO M for HSA and ESA, respectively. 

Albumin is responsible for the largest fraction of free sulfhydryl (Cys-34) in blood serum, and studies have shown that it is also the most reactive sulfhydryl. The chemical reactivity of Cys-34 is reported to have an unusually low p sH of 5 compared with 8.5 and 8.9 for cysteine and glutathione, respectively (Pedersen and Jacobsen, 1980). In most preparations of albumin, 30-35% of Cys-34 is occupied by cysteine or glutathione. Blocking of Cys-34 with cysteine, glutathione, or other chemicals such as A-idosuccinimide stabilizes albumin against dimer formation (Peters, 1985). Presumably, Cys-34 plays a direct or catalytic role in this process. 

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The precise nature of ligand binding has not been established in these complexes. 

The nature of the binding between the ligand and its complementary molecule... 

A majority of the host systems mentioned so far have been shown to encapsulate halide guests as a consequence of the symmetrical nature of their binding pockets, which readily adapt to the spherical symmetry of the halide ion. A study of the bis-tren macrobicyclic ligand 19-6H, however, has revealed that, in addition to accommodating halide anions, it is also able to encapsulate azide, N, within its cylindrical cavity. Solution stability constant measurements indicate that the host... 

Extensive research on albumin has led to an increasingly clear picture of ligand binding. The dye phenol red has been widely used as a model for the binding of natural ligands to proteins. Experimental results have shown that each molecule of albumin binds at least six molecules of phenol red. The presence of fatty acids such as decanoate, palmitate, stearate, and oleate... 

More recently, Perrella et al. (1992) have carried out a kinetic investigation on the binding of CO to Hb A. They find that the functional heterogeneity of the a and (3 chains of Hb A is dependent on the nature of allosteric effectors. In 0.1 M KC1 at pH 7 and 20°C, the 3 chains react about 1.5 times faster than the a chains with the first CO molecule. In the presence of IHP, the a chains react about 1.5 times faster than the 3 chains, but the overall rate of the first CO binding is unchanged. These data emphasize the point seen in much of the NMR work that the nature of ligands and allosteric effectors plays an important role in the functional properties of Hb A. 

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