Jaap Wilting

Location and characterization of the warfarin binding site of human serum albumin: A comparative study of two large fragments

The warfarin binding behaviour of a large tryptic fragment (residues 198-585 which comprise domains two and three) and of a large peptic fragment (residues 1-387 which comprise domains one and two) of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate and characterize the primary warfarin binding site. The induced ellipticity of the warfarin-peptic fragment complex turned out to be pH dependent. This pH dependence occurs in the region of the neutral-to-base transition of the albumin molecule. The induced ellipticity of the warfarin-tryptic fragment complex is pH independent. Difference CD-spectra showed that the peptic fragment and albumin have similar warfarin binding properties whereas the tryptic fragment has deviant warfarin binding properties. The equilibrium dialysis experiments showed that the affinity of warfarin to the peptic fragment and to albumin is practically the same whereas the affinity of warfarin to the tryptic fragment is a factor 2-8 lower than the affinity of warfarin to albumin. Our results indicate that the main part of the primary warfarin binding site is located in domain two of the albumin structure and that domain one plays an important role in the N-B transition of albumin.

Conformational changes in human serum albumin around the neutral pH from circular dichroic measurements

The molar ellipticity of the warfarin-albumin complex at 310 nm increases with pH from 6 to 9. This pH dependence runs parallel with that of the molar ellipticity of the albumin alone at 292 nm. The change in molar ellipicity with pH occurs in a smaller pH interval after addition of the physiological concentration of calcium ions. These findings give support to the assumption that the binding site for warfarin on the albumin molecule is affected by the neutral-to-base transition in the protein.

The role of albumin conformation in the binding of diazepam to human serum albumin

The effect of hydrogen, chloride and calcium ions on the binding of diazepam to human serum albumin has been studied by circular dichroism and equilibrium dialysis. In all cases the molar ellipticity of the diazepam-albumin complex increases with pH over the pH range 5 to 9. Under these conditions the free concentration of diazepam at a constant low drug to protein ratio decreases with pH. This free concentration is higher in the presence of chloride and calcium ions. With a two state conformational model for albumin it was shown, that the pH dependences of molar ellipticity of the diazepam-albumin complex and of the free concentration of diazepam are linked. It was demonstrated that the N-B transition of albumin is involved in the pH dependent binding of diazepam. The consequences of these findings for equilibrium dialysis procedures in determining free plasma levels of diazepam are discussed.

The uptake of the trypanocidal drug suramin in combination with low-density lipoproteins by Trypanosoma brucei and its possible mode of action.

In plasma, a significant part of suramin circulates in tight association with low-density lipoproteins (LDL). At therapeutically obtainable concentrations (100 microM) of suramin, about 85% of the total amount of the drug was bound to proteins, approximately 15% of which was bound to LDL. The molar ratio of suramin bound to LDL in serum was 7.5. The capacity of the high-affinity binding sites of LDL were 6.6 x 10(6) M-1, both in Tris buffer and in ultrafiltrate of serum. Suramin (100 microM) decreased the uptake of host LDL through receptor-mediated endocytosis by Trypanosoma brucei, with approximately 50%. LDL served as the only carrier for suramin uptake. Serum albumin, another important carrier for suramin in blood, was not able to promote suramin uptake, neither was delipidified plasma. The suramin taken up by T. brucei was recovered, in part, in the lysosomal fractions. It is suggested that deprivation of the parasite from cholesterol and phospholipids by an inhibition of the uptake of LDL, contributes to the mode of action of suramin, in addition to the many other effects that the drug may exert on the parasite. The toxic side-effects of suramin on the host are discussed in the light of its association with circulating lipoproteins.

Drug-binding and other physicochemical properties of a large tryptic and a large peptic fragment of human serum albumin

The diazepam-binding behaviour of a large tryptic and a large peptic fragment of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate the primary diazepam-binding site on the albumin molecule. The analytical set-up of the FPLC was used to find the optimum experimental conditions for isolating the fragments. Conventional columns with a 100-fold higher loading capacity than the analytical FPLC columns were used to isolate large amounts of the fragments. The isolation procedure for the tryptic fragment (45 kDa, domains two and three of the albumin structure) is described in this paper. The description of the isolation procedure for the peptic fragment (46 kDa, domains one and two of the albumin structure) is published elsewhere (Bos, O.J.M., Fischer, M.J.E., Wilting, J. and Janssen, L.H.M. (1988) J. Chromatogr. 424, 13-21). The induced ellipticity of the diazepam-fragment complexes as well as the affinity of diazepam to the fragments turned out to be pH dependent. This pH dependence occurs in the region of the neutral to base transition of the albumin molecule. Difference CD-spectra of the proteins showed that the tryptic fragment and albumin have similar diazepam-binding properties, whereas the peptic fragment has different diazepam-binding properties. This result is in line with our equilibrium dialysis experiments which showed that the affinity of diazepam to the tryptic fragment and to albumin is of the same order of magnitude, whereas the affinity of diazepam to the peptic fragment is several orders of magnitude lower. On the basis of these results, it can be concluded that the tryptic fragment contains the primary diazepam-binding site and the peptic fragment one or more secondary diazepam-binding sites. This means that at least the main part of the primary diazepam-binding site is located in domain three of the albumin structure.

Human serum albumin as an allosteric two-state protein. Evidence from effects of calcium and warfarin on proton binding behaviour

The proton binding of human serum albumin, and the influence on it of Ca2+ and warfarin (3-(alpha-acetonylbenzyl)-4-hydroxycoumarin), has been studied in the pH region 6 to 9, in order to get more information on the conformational change occurring in serum albumin around neutral pH, the so-called N-B transition. Some of the results for human serum albumin are compared with bovine serum albumin. A two-state model describing this transition is presented. In this model the two states are assumed to be the N state (found at low pH) and the B state (found at high pH). The ligand to be considered is the proton, having the highest affinity for the N conformation. An allosteric constant, L, governs the equilibrium between the two states. Both Ca2+ and warfarin can act as allosteric effectors, thereby increasing L. The model is used to describe results such as: (a) the cooperativity in proton binding, most clearly observable when Ca2+ is present, and the difficulty of measuring experimentally this cooperativity; (b) the different number of protons bound when Ca2+ is present or absent; (c) the fraction of protein found in one of the two conformations; (d) the correspondence between the increase of L due to addition of Ca2+ or warfarin, as predicted from model calculations, and the experimentally found increase of L.

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

Abstract 1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c . 2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent. 3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons ( k = 4 · 10 10 M −1 · s −1 at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10 7 M −1 · s −1 at 22 °C and pH 7.2. 4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase. 5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.

Mechanism by which warfarin binds to human serum albumin. Stopped-flow kinetic experiments with two large fragments of albumin.

In order to obtain information about the kinetics of the process by which warfarin binds to human serum albumin at a molecular level, we performed stopped-flow kinetic experiments on albumin and on a large peptic fragment (residues 1-387) and a large tryptic fragment (residues 198-585) of albumin. From these experiments it can be concluded that the first interaction between warfarin and the proteins is almost certainly diffusion-controlled and is dependent on the net charges of the reactants. The next step in the binding process involves the formation of an activated warfarin-protein complex, whereafter the final complex is formed. The warfarin-albumin complex forms more slowly than the warfarin-fragment complexes, because the formation is sterically hindered by the albumin structure. We think it very unlikely that albumin has an oblate ellipsoid structure; it is much more likely to have a U-shaped structure, where the domains make contact with each other. If this hypothesis is correct, then this indicates that the domains do not act independently of each other. The formation of the activated warfarin-albumin complex is further influenced by the conformational state of the albumin molecule, i.e. the N-B transition. The possible role of this N-B transition in albumin-mediated transport of drugs through cellular membranes is discussed.

Influence of pH on the binding of suramin to human serum albumin

The pH dependence of the binding of suramin to albumin has been studied by means of equilibrium dialysis and circular dichroism. Dialysis experiments have revealed that the association constants of the high and low affinity binding sites are strongly influenced by the pH. At pH 6.0 K1 = 1.4 x 10(6) M-1/n1 = 2.0 and K2 = 1.3 x 10(5) M-1/n2 = 1.0; at pH 9.2 K1 = 2.0 x 10(5) M-1/n1 = 2.0. At the high pH no low affinity sites could be demonstrated any more. The pH dependence of the induced ellipticity of the suramin-albumin complex at low molar drug-to-protein ratio r = 0.1 can be superimposed upon the neutral-to-base (N-B) transition of albumin alone. By means of the Linderstrøm-Lang equation for electrostatic interaction and a two-state model for the N-B transition of albumin, evidence is obtained of a link of the pH dependent binding behaviour of suramin to albumin and the neutral-to-base transition of albumin. The possible correlation of this link with transport processes of suramin in the body and with selective uptake of suramin in cells and parasites is discussed.

A comparative study of some physico-chemical properties of human serum albumin samples from different sources--I : Some physico-chemical properties of isoionic human serum albumin solutions

Human serum albumin samples from different sources were investigated. The fatty acid content of the albumin before and after deionization on a mixed bed ion-exchange column varied from sample to sample. When an albumin sample from one source was deionized under standard conditions the amount of fatty acid bound by the albumin was reduced to a reproducible amount. In samples from different sources, however, the amount bound varied considerably. Also the isoionic pH of the albumin varied from sample to sample. This variation could be attributed to the difference in the fatty acid content and the different number of titratable histidines and acid amino-acid residues in the albumin from different sources. It can be concluded from the specific conductance of these isoionic solutions that ions other than H-, OH and protein are effectively removed by a mixed bed ion-exchange column. The specific conductance of the albumin samples is directly related to the isoionic pH. Therefore, the isoionic pH and the specific conductance of the albumins reflect the heterogeneity of the albumin samples with respect to their primary structure and fatty acid content.