Growth of hemopoietic cells after incubation with VP 16-213

We examined the effect of various concentrations of VP 16-213 (25-125 microM/l, 2-h incubation on normal and complete remission bone marrow from patients with acute leukaemia and on leukaemic blasts. The maximal tolerated dose of the drug for normal bone marrow GM-CFC was between 75 and 100 microM/l whereas that for complete remission bone marrow was distinctly lower. More early stem cells measured by aid of LTBMC were more resistant in normal, but not in every remission bone marrow. We have to examine if these LTBMC results are influenced by a damaged microenvironment by using 2 stage LTBMC. Spontaneous leukaemic cells showed a different, sometimes lower sensitivity to VP 16-213 doses maximally tolerated by normal hemopoietic cells so that the VP 16-213 incubation must not be effective for every leukaemia.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.     

Horseradish peroxidase. XXXII. pH dependence of the oxidation of L-(-)-tyrosine by compound I.

The rate of oxidation of L-(-)-tyrosine by horseradish peroxidase compound 1 has been studied as a function of pH at 25 degrees C and ionic strength 0.11. Over the pH range of 3.20--11.23 major effects of three ionizations were observed. The pKa values of the phenolic (pKa = 10.10) and amino (pKa = 9.21) dissociations of tyrosine and a single enzyme ionization (pKa = 5.42) were determined from nonlinear least squares analysis of the log rate versus pH profile. It was noted that the less acidic form of the enzyme was most reactive; hence, the reaction is described as base catalyzed. The rate of tyrosine oxidation falls rapidly with the deprotonation of the phenolic group.     

Horseradish peroxidase binding to intestinal brush-border membranes of Cyprinus carpio. Identification of a putative receptor

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.     

Inhibition of the DNA catenation activity of type II topoisomerase by VP16-213 and VM26

Studies suggest that the anticancer drugs VP16-213 and VM26 produce cytotoxicity by inducing protein-associated DNA breakage in vivo through interaction with a yet unknown nuclear component. The effects of these drugs and their congeners on topoisomerase activities was investigated. VP16-213, VM26, and congeners active toward inducing DNA breaks also inhibited the catenation activity of eukaryote type II topoisomerase in vitro at very low drug concentrations. A structure-activity relationship was obtained for inhibition of catenation that parallels in vivo DNA breakage and cytotoxic activities. Type I topoisomerase activity was totally unaffected by these drugs.     

Horseradish peroxidase as a label of injured cells

Synopsis The present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusion test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H₂O₂ medium for the shortest possible period.Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.     

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.     

Analysis of the anticancer drugs VP 16-213 and VM 26 and their metabolites by high-performance liquid chromatography

A rapid and convenient high-performance liquid chromatographic procedure for the analysis of the clinically useful anticancer agents VP 16-213 and VM 26 is described. The drugs, which are semi-synthetic derivatives of the natural product podophyllotoxin, are extracted from plasma with chloroform. The extracts are evaporated to dryness, reconstituted in methanol, and chromatographed on a reversed-phase microparticle C₁₈ column using isocratic elution with a mixture of methanol—water (60:40). Each drug is used as the internal standard for the other. Quantitation to 500 ng/ml (0.85 nmole/ml) plasma is based on peak height ratios using UV detection at 254 nm. Patient plasma concentration vresus time data agree well with previously published data obtained using radiolabelled drug.Investigations into the nature of the hydroxy acid metabolite of VP 16-213, carried out using paired-ion chromatography with tetrabutylammonium bromide and fluorescence detection, are described. Also, a unique separation of VP 16-213 and a possible metabolite, the isomer, picro VP 16-213, is described.     

Horseradish peroxidase binding to intestinal brush‐border membranes of Cyprinus carpio. Identification of a putative receptor

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush‐border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K_d = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin‐type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3‐kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization. J. Cell. Biochem. 80:274–284, 2000. © 2000 Wiley‐Liss, Inc.     

Horseradish peroxidase: a modern view of a classic enzyme

Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.     

The oxidation of cytochrome c peroxidase by hydrogen peroxide. Characterization of products.

H2O2 reacts with cytochrome c peroxidase in a variety of ways. The initial reaction produces cytochrome c peroxidase Compound I. If more than a 10-fold excess of H2O2 is added to the enzyme, a portion of the H2O2 will react with Compound I to produce molecular oxygen. The remainder oxidizes the heme group and various amino acid residues in the protein. If less than a 10-fold excess of H2O2 is added to the enzyme, essentially all the H2O2 is utilized by oxidation of amino acid residues in the protein. The oxidation of the amino acid residues by H2O2 substantially modifies the reactivity of cytochrome c peroxidase. The modification of reactivity could be the direct result of amino acid oxidation or an indirect result caused by a perturbation of the protein structure at the active site. The products oxidized at pH 8 lose their ability to react with H2O2. The products oxidized at pH4 react with H2O2 but their reactivity toward Fe(CN)4-6 is substantially reduced.     

Determination of the molar absorptivities of phenothiazine cation radicals generated by oxidation with hydrogen peroxide/peroxidase.

Phenothiazines are used as antipsychotic drugs and as reagents to determine microamounts of hemoglobin in biological fluids and tissues. Several agents cause the oxidation of phenothiazines to chromophoric cation radicals, whose stability may be related with their biological action. Enzymes and proteins with peroxidase activity catalyze the oxidation by H2O2 of phenothiazines to their corresponding cation radicals, which suffer a nonenzymatic breakdown. The instability of these cation radicals makes the determination of their respective molar absorptivities very difficult. These properties, however, have been determined for a few phenothiazine cation radicals by cumbersome or unreliable procedures. In this paper a new method is proposed and applied to six different phenothiazines oxidized with H2O2/peroxidase. The method involves the stoichiometric exhaustion of H2O2, under assay conditions which yield a fast enzymatic formation of phenothiazine cation radicals and which slow down their nonenzymatic breakdown. This method may be useful for quantitative studies on the enzymatic activity and the reaction mechanism of the oxidation of a number of phenothiazines catalyzed by different types of peroxidase, as well as by proteins with peroxidase activity, such as hemoglobin.     

Horseradish peroxidase catalyzed hydroxylation of phenol: II. Kinetic model

A numeric kinetic model of the horseradish peroxidase catalyzed hydroxylation of phenol is proposed to complete the previous thermodynamic analysis. As previously stated, the basic role of HRP is to catalyze the production of DHF* radicals. These further form hydroxyl radicals that hydroxylate phenol via noncatalyzed reactions. The transient differential equations of the model are solved numerically. Several kinetic constants are adjusted to fit basic experimental data. This set of values is then kept constant to simulate additive experiments carried out under different conditions. Predictions of the model concerning the effects of HRP concentration, temperature variation, and presence of catalase and superoxide dismutase are consistent with the experimental results. The quantitative kinetic approach consequently fully confirmed the previous thermodynamic conclusions.     

Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase.

For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.