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Biomedical subjects

L H Janssen

Publications and source records attributed to L H Janssen.

At least 55 records · Page 3Linked to original sources
PubMed1989

Is hemoglobin a catalyst for sulfoxidation of chlorpromazine? An investigation with isolated purified hemoglobin and hemoglobin in monooxygenase and peroxidase mimicking systems.

The possible role of hemoglobin in the sulfoxidation of chlorpromazine is still a controversial subject. Therefore this sulfoxidation was investigated with purified oxyhemoglobin and methemoglobin under various conditions: (i) in phosphate buffer pH 6.5; (ii) in monooxygenase mimicking systems with electron donors like ascorbic acid and NADPH, the last, with and without an electron carrier like methylene blue and cytochrome c reductase; (iii) in the presence of H2O2. Only in the presence of H2O2 chlorpromazine was converted into chlorpromazine sulfoxide in a considerable amount. This so-called peroxidase activity of hemoglobin appeared not to be based on a Fenton-type reaction. An oxidized reactive form of hemoglobin (i.e. ferrylhemoglobin) is responsible for the sulfoxidation. In the other systems only with ascorbic acid some chlorpromazine sulfoxide was produced. This is probably due to the production of H2O2 and the subsequent peroxidase activity of hemoglobin. Chlorpromazine enhanced the autoxidation of oxyhemoglobin, without being transformed itself.

Chlorpromazine↗
PubMed1989

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.

Circular Dichroism↗
PubMed1989

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.

Humans↗
PubMed1989

The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin.

In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.

Binding Sites↗
PubMed1989

DNA alkylation and formation of DNA interstrand cross-links by potential antitumour 2,5-bis(1-aziridinyl)-1,4-benzoquinones.

A series of 3,6-substituted 2,5-bis(1-aziridinyl)-1,4-benzoquinone derivatives was shown to alkylate calf thymus DNA and to form DNA interstrand cross-links. Alkylation and cross-link formation were enhanced after electrochemical reduction of the compounds and increased with lower pH in the pH range from 4.5 to 8.0. Reduction especially shifts the pH at which cross-linking and alkylation occurs to higher values, which are more physiologically relevant. This shift is probably caused by the increase in pKa value of the aziridine ring after reduction of the quinone moiety. The inactivation of single-stranded bacteriophage M13mp19 DNA to form phages in an E. coli host, by the 3,6-unsubstituted parent compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone (TW13) was dependent upon reduction and pH in a similar way as was alkylation. The compound in our series with the least bulky, 3,6-substituents, TW13, caused a high amount of cross-link formation. Compounds with methyl-substituted aziridine rings showed low cross-linking ability. Our results support the concept that the protonated reduced compound is the reactive species that alkylates DNA, and that steric factors play an important role in the reactivity towards DNA. A correlation is observed between the ability to induce DNA interstrand cross-links and inactivation of M13mp19 bacteriophage DNA. Cross-link formation was also demonstrated in E. coli K12 cells, where the compounds are reduced endogenously by bacterial reductases.

Alkylation↗
PubMed1988

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.

Binding Sites↗
PubMed1988

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.

Circular Dichroism↗
PubMed1988

The fatty-acid-induced conformational states of human serum albumin investigated by means of multiple co-binding of protons and oleic acid.

The binding of oleic acid to human serum albumin causes progressive changes in (a) the pK of some amino acid residues, as detected by pH-stat titration and (b) the induced molar ellipticities of albumin-bound drugs (diazepam and oxyphenbutazone), as measured by c.d. It is concluded that albumin undergoes several conformational transitions as the amount of oleic acid bound increases from 0 to about 9 molecules/molecule of protein. At least three different conformations of the protein seem to be involved. These conformations can be linked with the three classes of oleic acid-binding sites on albumin.

Binding Sites↗
PubMed1988

Procedure for the isolation of a large peptic fragment of human serum albumin.

For a thorough investigation of the drug-binding behaviour and other physicochemical properties of human serum albumin, one needs large amounts of specific fragments of albumin. Such fragments were obtained by careful proteolysis of the native protein with pepsin at pH 3.70. The fast protein liquid chromatographic technique was used to find the optimum experimental conditions for the separation of the fragments. By means of anion-exchange chromatography, chromatofocusing and gel permeation, it was possible to obtain a large fragment with a relative molecular mass of 46,000. The fragment could be assigned to segment 1-387 and therefore consists of domains 1 and 2 of the albumin structure. A 1-g amount of albumin produced 50 mg of a fragment that was 98% homogeneous.

Amino Acids↗
PubMed1988

Potential anti-tumour 2,5-bis (1-aziridinyl)-1,4-benzoquinones: relationship between cytotoxicity against DNA-repair deficient E. coli, inactivation of bacteriophage M13mp19, and in vitro and in vivo anti-tumour activity.

A series of reductively activated 3,6 substituted 2,5-bis(aziridinyl)benzoquinones, designed as potential anti-tumour agents, was shown to interact with DNA. This was assayed by studying the colony forming ability of DNA-repair deficient E. coli K12 and the inactivation of bacteriophage M13mp19. The activities of the compounds as measured in the two assays show a good correlation. Reduction of the quinone compounds is essential for activity in the two assays. Reduction activates opening of the aziridine ring and formation of an alkylating species. The results suggest that steric factors are important in DNA adduct formation. Methylation of the aziridine rings decreases the activity. Bulky 3,6-substitutes also decrease the activity. No correlation is found between above-mentioned assays for DNA modification and in vivo activity towards tumour cells. It is possible that, as well as DNA, other cellular targets are involved. A correlation is observed between the DNA modifying activity and toxicity against L1210 cells in vitro. The relative high activity against L1210 cells of bromine substituted compounds is possibly related to alternative mechanisms in which bromine acts as an efficient leaving group.

Animals↗
PubMed1986

A proton nuclear magnetic resonance study of human serum albumin in the neutral pH region.

By means of 500 MHz 1H-NMR spectroscopy detailed information has been obtained about the proton titration behaviour of the histidine residues of human serum albumin under various experimental conditions. The approximate number of His C2 protons resolved varied from 11 at pH* 9.0 to 16 at pH* 5.5. The latter is the same as the number of histidine residues reported to be present in the molecule. The symbol pH* stands for the meter readings uncorrected for the deuterium isotope effect. Whereas some of the NMR signals remained visible over the entire pH* range examined (5-9), others, having pH*-dependent resonance intensities, were observed in part of the pH* range only. The latter effect is indicative of an alteration in the histidine environment in the course of pH* titration. It is thought that the so-called N-B conformational transition, occurring in the neutral pH region, is responsible for this alteration. The spectral effects observed upon the addition of Ca2+ can be interpreted in terms of a downward pK* shift for a number of histidine residues and a concomitant downward shift in the midpoint pH* of the N-B transition. This corroborates the results of previous investigations on this matter.

Calcium Chloride↗
PubMed1986

The influence of Ca2+ on the turbidity of DPPC-DMPA vesicles within the temperature range of the phase transition.

The influence of the addition of Ca2+ on the phase behaviour of vesicles, composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidic acid (DMPA) in a ratio of 4 to 1, has been investigated by means of turbidity measurements. As expected one single phase transition for the mixed phospholipids was observed in the absence of Ca2+. Passing through the temperature range of this transition after the addition of Ca2+, conditions appeared to favor fusion of the vesicles. A possible reason for this is that during the transition Ca2+ may permeate through the vesicle membranes and gain access to the inside DMPA binding sites. Therefore it is not unambiguously possible to determine phase transition temperatures from the turbidity changes that occur under these conditions. However, when within the temperature range of the phase transition of the mixed phospholipids the influence of Ca2+ addition to the vesicles was recorded isothermally, at each temperature separately, the final plot of turbidity versus temperature turned out to be far less confused by fusion events and adopted the form of two separate phase transitions. The temperatures at which these two transitions occur closely resemble the phase transition temperatures that may be observed in the absence of Ca2+ for DMPA and DPPC alone, 39 degrees C and 43 degrees C respectively. The results of this study suggest that when Ca2+ has only access to the outside of the vesicle membranes it may segregate the neutral and the acidic phospholipids into separate domains, both domains adopting their proper phase condition at the actual temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1985

The pH dependence of the thermodynamics of the interaction of diazepam with human serum albumin.

The heat effect associated with the binding of diazepam to human serum albumin has been measured from pH 5.5 to 9.5 using flow microcalorimetry. The number of protons released upon the binding of one molecule of diazepam to one molecule of albumin was determined from acid-base titration experiments. In addition, the heat associated with the combination of protons with serum albumin was measured calorimetrically. The results of the experiments using these two techniques were used to correct the measured heat of binding and to obtain what will be called here delta H0obs values. These delta H0obs values are strongly pH-dependent: up to pH 7.5 a value of -24.3 (+/- 0.5) kJ X mol-1 is found, whereas at pH 9.5 the value is -44.3 (+/- 1.2). This pH dependence is different from the pH dependence of the induced CD signal of the albumin-diazepam complex. A model is presented in which the pH dependence of delta H0obs is explained. This model takes into account the N-B (neutral to base) conformational change in albumin around neutral pH. Parameters in this model are KN and KB (representing the affinity of diazepam for albumin in the N and B conformation, respectively), delta H0N and delta H0B which are the corresponding standard enthalpy changes, f which represents the fraction of protein in the B conformation, and delta H0(N-B) which represents the heat associated with the N-B transition.

Diazepam↗
PubMed1985

Evidence for the fatty acid-induced heterogeneity of the N and B conformations of human serum albumin.

The influence of oleic acid on the interaction between albumin and warfarin, oxyphenbutazone or diazepam has been studied by circular dichroism and equilibrium dialysis. The pH dependences of the molar ellipticity of the drug-albumin complexes and of the free fraction of drug are completely changed by the presence of oleic acid. This phenomenon is attributed to an oleic acid-induced conformational change in both the neutral (N) and the basic (B) conformation of albumin, a change to which the warfarin-oxyphenbutazone binding area and the diazepam binding site is sensitive. The oleic acid-induced conformational states of albumin, the so-called N* and B* conformations, show binding properties that are different from the binding properties of the N and B conformations.

Circular Dichroism↗
PubMed1985

A study on the allosteric interaction between the major binding sites of human serum albumin using microcalorimetry.

The binding of warfarin and oxyphenbutazone to albumin has been studied at pH 6.8 and pH 9.2 by measuring the heat of binding of these ligands to their high-affinity binding sites on albumin (delta Ho'1). The -delta Ho'1 values for the binding of warfarin at pH 6.8 and 9.2 and oxyphenbutazone at pH 6.8 and 9.2 were found to be 16.9(+/- 0.6), 28.8(+/- 0.6), 10.5(+/- 0.4) and 17.4(+/- 0.6) kJmol-1, respectively. The Gibbs energies (delta Go'1) corresponding to these delta Ho'1 values cover a much smaller range. The pH dependences of delta Go'1 and delta Ho'1 are explained in terms of pK shifts in the albumin upon binding warfarin or oxyphenbutazone. Diazepam, which binds to a site on albumin which is different from the warfarin-oxyphenbutazone binding site, increases - delta Ho'1 for the binding of warfarin and oxyphenbutazone to albumin at pH 6.8, but it does not influence the -delta Ho'1 at pH 9.2. This phenomenon may be attributed to an allosteric interaction between the diazepam binding site and the warfarin binding site. This allosteric interaction must have its origin in a phenomenon other than the N-B transition.

Binding Sites↗