Lambert H.M. Janssen

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.

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.

Oxidation of chlorpromazine by methemoglobin in the presence of hydrogen peroxide. Formation of chlorpromazine radical cation and its covalent binding to methemoglobin

The oxidation of chlorpromazine by methemoglobin plus H2O2 has been studied. The transient formation of the chlorpromazine radical cation in this reaction has been demonstrated by light absorption measurements. Under the experimental conditions complete conversion of chlorpromazine yields approximately 60% chlorpromazine sulfoxide. From studies with 3H-labeled chlorpromazine it appears that the remaining 40% is covalently bound to apohemoglobin. Upon reaction of methemoglobin with H2O2 a stable ferrylhemoglobin is formed. This ferrylhemoglobin is not the reactive species, which accepts the chlorpromazine electron, as its presence is not sufficient to induce chlorpromazine oxidation. For this the presence of H2O2 is a prerequisite. This indicates that a transient species in the formation of the stable ferrylhemoglobin is involved, whether this is a compound I analogue or a ferrylhemoglobin with a free radical on one of the apoprotein residues. Exposition of methemoglobin to H2O2 denatures hemoglobin and induces protein-heme crosslinks, as appears from changes in the visible absorption spectrum and heme retention by the protein after methyl ethyl ketone extraction. Reaction with CPZ partly protects against denaturation and crosslinking.

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.

Kinetic evaluation of the oxidation of phenothiazine derivatives by methemoglobin and horseradish peroxidase in the presence of hydrogen peroxide. Implications for the reaction mechanisms.

The oxidation of ten 2-substituted 10-(3-(dimethylamino)propyl) phenothiazines (PHs) by methemoglobin (metHb) and horseradish peroxidase (HRP) in the presence of H2O2 was kinetically analysed based on an enzymic-chemical second-order reaction with substrate regeneration: PHs are oxidized enzymatically to their radical cations (PH+) which subsequently, in a second order reaction, react further to parent compound and PH-sulfoxide (PHSO). The enzymic reaction rate can be obtained from the accumulation curves of both radical cation formation and sulfoxide formation. In the case of chlorpromazine and promazine both methods gave similar reaction rates. The rate constant of PH+. decay could also be determined from the radical concentrations of their radicals. The rate constant of reaction of PHs with HRP compound II was also analysed. The logarithm of this rate constant correlated well with the Hammett sigma para and the Swain and Lupton F and R substituent constants, whereas no correlation with hydrophobic and steric parameters was found. This indicates that the interaction of PH with the porphyrin ring, which is the active site of HRP, is predominantly under electronic control. In the case of catalysis by hemoglobin (Hb), the formation of the reactive Hb form, ferry1Hb with a protein radical, appeared to be rate limiting in the oxidation of PHs by metHb-H2O2. Differences in the conversion rates of various PHs can be explained by a competition between their electron transfer reaction to the protein radical and the denaturation reaction(s) involving the protein radical. Our results confirm our earlier observation that the mechanism of oxidation by metHb-H2O2 differs from that of the classical peroxidases. In the former case, electron transfer from PH occurs most likely to a tyrosine residue on the globin part, whilst in the latter case electron transfer to the porphyrin moiety takes place.

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.

Inhibition of mediator release in RBL-2H3 cells by some H1-antagonist derived anti-allergic drugs: Relation to lipophilicity and membrane effects

In a model for mucosal mast cells (RBL-2H3 cells) a set H1-antagonist derived anti-allergic drugs containing a diphenylmethyl piperazinyl moiety was examined for their ability to inhibit release of the mediatorβ-hexosaminidase. Cells were activated with antigen or the calcium ionophore A23187, whether or not in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Oxatomide, hydroxyzine and cetirizine inhibit the antigen inducedβ-hexosaminidase release. The release triggered by A23187, whether or not in combination with TPA is hardly influenced by the compounds. A biphasic dependence of the inhibition of exocytosis in RBL cells on lipophilicity is observed with the optimum at log P is 5–6. The extremely lipophilic compounds meclozine and buclizine are not active in this model. pH dependence of the effect of the drugs shows that especially the uncharged species are active in inhibiting exocytosis. The investigated compounds show an effect on phase transitions in L-α-phosphatidylcholine dipalmitoyl liposomes as assayed with differential scanning calorimetry (DSC). For the less extremely lipophilic compounds the induced changes in the phospholipid membranes increased with lipophilicity. The relation between structural features of the drug and the interaction with phospholipids is discussed in view of the DSC results. We conclude that location of the active drugs at the membrane or the membrane/protein interface is important for the inhibiting activity on exocytosis. This could affect several membrane related processes, which are abundant in the early phases of the IgE-mediated signal transduction process.

Indoloquinone EO9: DNA interstrand cross-linking upon reduction by DT-diaphorase or xanthine oxidase.

We report DNA interstrand cross-linking caused by the anti-tumour indoloquinone EO9 following reductive activation with purified rat liver DT-diaphorase or xanthine oxidase. Reduction was a necessary event for cross-linking to occur. DNA cross-link formation by EO9 following DT-diaphorase reduction was completely inhibited by addition 10 microM dicoumarol, whereas only a minor effect of dicoumarol on xanthine oxidase-mediated DNA cross-linking by EO9 was observed. DNA cross-linking was pH dependent, with increasing cross-link formation from pH 5.5 to 7.0 for both DT-diaphorase and xanthine oxidase mediated reactions. Also, conversion of EO9 upon reduction was pH dependent. However, in contrast to DNA cross-linking, conversion rates of EO9 decreased at higher pH. EO9 was shown to be more efficient in DNA cross-linking than mitomycin C under identical conditions, using both DT-diaphorase and xanthine oxidase reductive activation at pH 5.5 and 7.0. This study indicates that the anti-tumour activity of EO9 may be at least partly mediated by interstrand DNA cross-link formation, and that various reducing enzymes may be important for activation of EO9 in vitro and in vivo.

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.