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Biomedical subjects

B B Hasinoff

Publications and source records attributed to B B Hasinoff.

At least 73 records · Page 4Linked to original sources
PubMed1989

The interaction of the cardioprotective agent ICRF-187 [+)-1,2-bis(3,5-dioxopiperazinyl-1-yL)propane); its hydrolysis product (ICRF-198); and other chelating agents with the Fe(III) and Cu(II) complexes of adriamycin.

Membrane-permeable ICRF-187 [+]-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane) has shown promise as a cardioprotective agent against adriamycin-induced cardiotoxicity. ICRF-187 may act through its rings-opened hydrolysis product (ICRF-198), which has an EDTA-type structure and, likewise, strongly binds metal ions. The reactions of these compounds with Fe3+-adriamycin and Cu2+-adriamycin complexes were examined. ICRF-198 quickly and completely removed both Fe3+ and Cu2+ from their complexes with adriamycin. ICRF-187 also reacted directly, but more slowly, with Fe3+-adriamycin to remove Fe3+ from the complex. This reaction was first order in ICRF-187 and Fe3+-adriamycin and yielded a second order rate constant of 123 M-1 min-1. Metal ion-complex promoted hydrolysis may thus contribute to the in vivo hydrolysis of ICRF-187 to its metal ion-chelating active rings-opened form. Both ICRF-187 and ICRF-198 were very effective in preventing the Fe3+-adriamycin induced inactivation of the cytochrome c oxidase activity of submitochondrial particles. A number of other chelating agents (desferal; penicillamine; DTPA; EDTA; TPEN; bathophenanthroline sulfonic acid; 2,2'-bipyridine; 1.10-phenanthroline, glutathione and 2-mercaptoethanol) were also examined for their ability to remove Fe3+ and Cu2+ from their complexes with adriamycin.

Cardiotonic Agents↗
PubMed1989

Self-reduction of the iron(III)-doxorubicin complex.

The Fe3(+)-doxorubicin complex undergoes reactions that suggest that the complex self-reduces to a ferrous oxidized-doxorubicin free radical species. The Fe3(+)-doxorubicin system is observed to reduce ferricytochrome c, consume O2 and react with 2,2'-bipyridine. Bipyridine acts as a "ferrous ion scavenger" as it reacts with the ferrous ion produced by Fe3(+)-doxorubicin self-reduction. In the absence of O2, a ferrous doxorubicin complex accumulates. In the presence of oxygen, Fe2+ recycles back to Fe3+. The rates of these reactions were measured and the Fe3(+)-doxorubicin self-reduction was determined to be the rate-determining step. The Fe3(+)-doxorubicin induced inactivation of cytochrome c oxidase and NADH cytochrome c reductase on beef heart submitochondrial particles occurs at a rate similar to Fe3(+)-doxorubicin self-reduction. Thus the rate at which damage to these mitochondrial enzymes occurs may be controlled by a nonenzymatic Fe3(+)-doxorubicin self-reduction.

Aerobiosis↗
PubMed1989

The Adriamycin (doxorubicin)-induced inactivation of cytochrome c oxidase depends on the presence of iron or copper.

1. It was confirmed that Adriamycin (doxorubicin) inactivates cytochrome c oxidase upon incubation. However, further investigation shows that this inactivation is strongly dependent upon the presence of Fe3+ and Cu2+. Trace amounts of these transition metal ions, present in phosphate and Tris buffers, bind strongly to the Adriamycin and the complex formed is responsible for the inactivation of cytochrome c oxidase. No Adriamycin-induced inactivation of cytochrome c oxidase occurred in the presence of EDTA or in phosphate buffers purified on a cation exchange column to remove trace metals. 2. The metal ion-induced inactivation of cytochrome c oxidase by Adriamycin results in significant decreases in both the maximum velocity and the Michaelis constant. The degree of inactivation is strongly dependent on the Fe3+ concentration. 3. Cardiolipin partially protects against cytochrome c oxidase inactivation, presumably by binding to the cytochrome c oxidase, whereas catalase or superoxide dismutase partially protect by scavenging damaging reactive oxygen species generated within a Fe3+-Adriamycin-enzyme complex.

Animals↗
PubMed1988

Adriamycin and its iron(III) and copper(II) complexes. Glutathione-induced dissociation; cytochrome c oxidase inactivation and protection; binding to cardiolipin.

Some reactions of adriamycin (doxorubicin) and its Fe3+ and Cu2+ complexes were investigated with a view to understanding the mechanisms by which metal ion-adriamycin complexes damage cellular components. The ability of adriamycin in the presence of Cu2+ to inactivate the mitochondrial enzyme cytochrome c oxidase was effectively prevented by physiologic levels of glutathione. This result is explained by the observation that glutathione reacts with the Cu2+-adriamycin complex to produce free adriamycin. As sulfhydryl compounds are, in contrast, known to promote Fe3+-adriamycin-induced damage to cellular components, these results suggest that the response of a metal ion-adriamycin system to the presence of sulfhydryl compounds may be indicative of whether or not Cu2+-adriamycin is the damaging species. The partition of adriamycin into the octanol phase of an octanol-water two-phase system was greatly enhanced by the presence of cardiolipin. This result can be explained by the formation of a strong adriamycin-cardiolipin complex in the octanol phase which is one-half formed at an adriamycin concentration of 6 microM.

Cardiolipins↗
PubMed1988

The iron(III)-adriamycin complex inhibits cytochrome c oxidase before its inactivation.

Cytochrome c oxidase was found to be competitively inhibited by a complex formed between Fe3+ and the cardiotoxic antitumour drug adriamycin (doxorubicin) with an inhibition constant, Ki, of 12 microM. This competitive inhibition precedes the slower Fe3+-adriamycin induced inactivation of cytochrome c oxidase. In strong contrast with this result, free adriamycin was not observed to either inhibit or inactivate cytochrome c oxidase (Ki greater than 3 mM). Since, typically, polycations are known to inhibit cytochrome c oxidase, the competitive inhibition displayed by the Fe3+-adriamycin complex may also result from its polycationic character. Cytochrome c oxidase was also inhibited by pentan-1-ol (Ki 13 mM), and kinetic studies carried out in the presence of both inhibitors demonstrated that the Fe3+-adriamycin complex and pentan-1-ol are mutually exclusive inhibitors of cytochrome c oxidase. The inhibitor pentan-1-ol was also effective in preventing the slow inactivation of cytochrome c oxidase induced by Fe3+-adriamycin, presumably by blocking its binding to the enzyme. It is postulated that the slow inactivation of cytochrome c oxidase occurs when reactive radical species are produced while the Fe3+-adriamycin is complexed to cytochrome c oxidase in an enzyme-inhibitor complex. The Fe3+-adriamycin-induced inactivation of cytochrome c oxidase may be, in part, responsible for the cardiotoxicity of adriamycin.

Animals↗
PubMed1987

The kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome c oxidase in solvents of increased viscosity are partially diffusion controlled.

The steady-state spectrophotometrically determined initial velocity kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome c oxidase were examined for effects of diffusion control in solvents of increased viscosity. Both glycerol/water and sucrose/water proved unsatisfactory as viscosogens due to weak competitive inhibition (Ki values of 2.6 M and 1.6 M, respectively). However, polyethylene glycol (PEG) was satisfactory as a viscosogen. The measured diffusion coefficient of ferrocytochrome c in PEG/water was shown to follow closely the Stokes-Einstein equation. In PEG/water mixtures at high ionic strength the minimum association rate constant (kmin = Vmax/(Km[EO]) is partly diffusion controlled with contributions from diffusion control and chemical activation control being about equal at 5 mPa X s, a viscosity that may be physiologically relevant. This finding can be interpreted to mean that cytochrome c oxidase is an enzyme that has evolved to approach its maximum efficiency. The steady-state kinetics were also examined at low ionic strength where multiphasic kinetics are exhibited. The effect of increased viscosity was exhibited over the whole experimentally accessible region indicating that there are effects due to diffusion control on both the high-affinity and low-affinity binding of ferrocytochrome c. Several models for diffusion control were examined and a comparison is made with other diffusion-controlled reactions of proteins.

Aerobiosis↗
PubMed1987

The association reaction of yeast alcohol dehydrogenase with coenzyme is partly diffusion-controlled in solvents of increased viscosity.

The steady-state kinetics of the yeast and liver alcohol dehydrogenase catalyzed reduction of aldehydes were examined in solvent mixtures of increased viscosity. This was done to investigate the effects of diffusion control on the fast association of NADH with the enzymes. Both glycerol and sucrose were unsatisfactory as viscosogens, as they inhibited the enzyme, but poly(ethylene glycol)/water mixtures were satisfactory. The 5-fold faster reaction of yeast alcohol dehydrogenase with NADH is partly diffusion controlled, whereas the slower liver alcohol dehydrogenase reaction showed no diffusion effects. These results are consistent with a yeast alcohol dehydrogenase active site that has relatively little steric hindrance to NADH binding. It is estimated that contributions to this association reaction from diffusion control and chemical activation control are equal at a solvent viscosity of 10 cP. Thus, under physiological conditions of increased viscosity the NADH association may be significantly affected by diffusion effects. In order to estimate accurately the maximum diffusion-controlled rate constant from diffusion theory, the diffusion coefficients of NADH were measured in poly(ethylene glycol)/water mixtures and were found to vary inversely as the solvent viscosity raised to the power of 0.5. The non-Stokesian behaviour of molecules as large as NADH in polymer/water mixtures may be a serious limitation to the routine use of poly(ethylene glycol) as a viscosogen for diffusion studies.

Alcohol Dehydrogenase↗
PubMed1986

Pig heart fumarase really does exhibit negative kinetic co-operativity at a constant ionic strength.

The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.

Animals↗
PubMed1984

Kinetics of carbonic anhydrase catalysis in solvents of increased viscosity: a partially diffusion-controlled reaction.

Steady-state kinetic studies of the bovine carbonic anhydrase B-catalyzed hydration of CO2, dehydration of HCO3-, and hydrolysis of p-nitrophenylacetate were made in glycerol/water solvents of increased viscosity in order that the effect of diffusion-control on the substrate association reactions could be determined. The minimum association rate constants (kmin = V/(Km[E0])) were obtained at low substrate concentrations. The esterase activity did not depend upon the solvent viscosity. However, both the CO2 hydration and HCO3- dehydration reactions depended upon the solvent viscosity consistent with partial diffusion control. Thus both chemical activation and diffusion control processes contribute to the observed kmin. In low-viscosity aqueous solutions both hydration and dehydration are largely controlled by chemical activation. However, at higher viscosities, equal to that found in the interior of the erythrocyte, both reactions are largely diffusion controlled. This result can be interpreted to mean that carbonic anhydrase is a highly evolved enzyme that has approached its maximum efficiency. The extent of diffusion control observed rules out H2CO3 as a significant reactant with the enzyme. Several models that yield minimum steric requirements for access of substrate to the active site are examined. Minimum steric constraints are less for the smaller CO2. The slower esterase reaction is not influenced by diffusion.

Animals↗
PubMed1983

Pulse radiolysis kinetics of the reaction of hydrated electrons with ferric-, ferrous-, protoporphyrin IX- and apo-myoglobin.

The kinetics of the reaction of hydrated electron (e-aq) with ferric-, ferrous-, metal-free protoporphyrin IX-and apo-myoglobin have been studied by pulse radiolysis so that a direct kinetic measure of the relative reactivities of the heme and the protein part of myoglobin can be made. The second-order association rate constant with ferric Mb is about 3-times that for ApoMb, while ferric Mb, ferrous Mb and protoporphyrin IX-Mb all react at about the same rate, indicating that it is mainly the porphyrin that is the electron-attracting site. The magnitude of the rate constants (8-25 nM-1 X S-1) indicates that the encounter of e-aq with the protein is almost certainly diffusion-controlled. The initial encounter is probably followed by electron migration along parallel paths to the heme and most likely several of the 12 histidine residues. The heme competes very effectively (approx. 70%) with these other sites. The kinetically measured reduction yield of heme is consistent with that found spectrally, indicating that a histidine radical on the protein does not effectively transfer an electron intramolecularly to the heme. The spectral changes found upon the completion of the fast reaction (approx. 40 microseconds) for protoporphyrin IX-Mb and ferrous Mb are consistent with the formation of a porphyrin anion radical. For ApoMb the spectral changes are consistent with the formation of a histidine free radical.

Animals↗
PubMed1983

Viscosity dependence of the kinetics of the diffusion-controlled reaction of carbon monoxide with the separated alpha and beta chains of hemoglobin.

The kinetics of the recombination reaction of carbon monoxide with the isolated alpha and beta chains of human hemoglobin have been examined by laser flash photolysis in glycerol-water as a function of temperature and solvent viscosity. The second-order recombination rate constant is inversely proportional to viscosity raised to the 0.5 power--paralleling that predicted for the CO diffusion coefficient. This viscosity exponent is independent of the protein. These results are consistent with the reaction kinetics being essentially diffusion controlled in the high viscosity glycerol-water. For the alpha and beta chains, respectively, the diffusion-controlled rate constant is 0.003 and 0.002 of that predicted from the simple von Smoluchowski model based on the diffusion coefficients and molecular sizes of uniformly reactive spherical molecules. Several models incorporating steric requirements are used to rationalize the results. These models indicate that steric requirements for reaction in the diffusion-controlled limits are not greatly different in the alpha and beta chains and are only slightly less stringent than for myoglobin.

Carbon Monoxide↗
PubMed1982

Viscosity dependence of the kinetics of the diffusion-controlled reaction of carbon monoxide and myoglobin.

The kinetics of the reaction of CO with myoglobin have been studied by laser flash photolysis in glycerol-water as a function of solvent viscosity and temperature. At high viscosities and low temperatures the second-order rate constant is inversely proportional to the viscosity raised to approximately the 0.5 power. This result parallels the behavior of the oxygen diffusion coefficient in glycerol-water. It is concluded that the reaction kinetics in high viscosity glycerol-water are largely diffusion controlled. At higher temperatures, though, the effect of simultaneous chemical activation control of the reaction is observed. The diffusion-controlled rate constant is 1.4 X 10(-3) of that predicted from simple von Smoluchowski theory based on diffusion coefficients and molecular radii. Several models with steric requirements for diffusion-controlled reactions are examined.

Carbon Monoxide↗
PubMed1982

Kinetics of acetylthiocholine binding to electric eel acetylcholinesterase in glycerol/water solvents of increased viscosity. Evidence for a diffusion-controlled reaction.

Steady-state kinetic studies were made on the very efficient enzyme hydrolysis of acetylthiocholine by electric eel acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) in glycerol/water solvents of increased viscosity. Determinations of the very fast minimum substrate association rate constants kmin, (2 . 10(8) M-1 . s-1 at I approximately 0.1 M and 25 degrees C) from the Michaelis parameters, V/(Km[E0]), were made at low substrate concentrations in order to obtain kmin directly. kmin was shown to be strongly dependent upon viscosity, which is characteristic of a diffusion-controlled reaction. kmin is as large or larger than plausible models for a simple diffusion-controlled reaction between a charged enzyme and substrate would suggest. Enhancement of the diffusion-controlled reaction through nonspecific binding of substrate to the highly negatively charged acetylcholinesterase followed by two-dimensional surface diffusion in a random walk to the active site may be a factor in this enzyme mechanism. Evidence for this comes from the viscosity dependence of kmin. Using the surface diffusion model it is estimated that the binding-site target area on acetylcholinesterase is effectively increased a minimum of 8-fold.

Acetylcholinesterase↗
PubMed1982

Kinetics of the reaction of carbon monoxide and oxygen with T-state hemoglobin in human red blood cell suspensions studied by dye laser flash photolysis.

The reaction kinetics of carbon monoxide and oxygen binding to slow reacting T-state "deoxy" hemoglobin (HbT) in human red blood cell (RBC) suspensions were studied with a 300-ns pulse width dye laser to photodissociate either HbCO or HbO2. The rate constants were determined using only the first 10% of the slow HbT reaction, as at longer times diffusion of ligand away from the RBC seriously affects the kinetics. The rate constants measured are similar to those in solution, indicating that the different physical environment in the RBC has little effect on the Hb kinetics.

Carbon Monoxide↗
PubMed1981

Kinetics of carbon monoxide and oxygen binding to hemoglobin in human red blood cell suspensions studied by laser flash photolysis.

The reaction kinetics of the binding of CO and O2 to hemoglobin (Hb) in human red blood cell (RBC) suspensions have been examined using a 300 ns dye laser to photodissociate HbCO or HbO2. Fast (halftime approximately or equal to 10 microsecond) and slow (approximately or equal to 5 ms) processes were seen after photolysis. The results indicate that neither the rate constants nor the activation energies for the binding of CO to the fast reacting form of Hb in the RBC are significantly different from that measured in solution in spite of the different environments. Rate constants determined for O2 binding in RBC were intermediate between rates observed for reaction with fast ans slow reacting forms of Hb and probably consist of contributions from each. The slow recombination of CO and O2 probably has contributions both from reaction with slow reacting forms of Hb and from ligand that had diffused away from the RBC after photolysis.

Carboxyhemoglobin↗