A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Hydroxyl free radical production in iron-cysteine solutions and protection by zinc

Hydroxyl radicals (OH^?) can be formed on incubation of an oxygenated solution of ferrous sulphate and cysteine. This has been demonstrated by esr using the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide), catalase, and the radical scavengers ethanol and propan-2-ol. Hydroxyl radicals are not formed when excess zinc sulphate is present. These results provide support for the pro-oxidant action of iron and cysteine and a possible protective role for zinc.     

Kinetic studies on the cupric ion oxidation of sheep hemoglobin

The oxidation of sheep hemoglobin, in both the oxygenated and deoxygenated forms, by cuprous ions have been studied by spectrophotometric and stopped-flow techniques. Mixing of both the oxy and deoxy forms with excess Cu²⁺ leads to the rapid oxidation of the iron atoms of all four of the hem groups of the tetrameric protein, followed by the slow formation of hemichromes (low spin Fe^III forms of hemoglobin). Stopped-flow studies show that the oxidations follow simple monophasic kinetics with second-order rate constants of 65 and 310 M^?1 sec^?1 for the oxy and deoxy forms, respectively. Variable temperature studies yield Arrhenius activation energies of 43 for the oxy form and 113 kJ mole^?1 for the deoxy form. For each form of the protein the activation energy is very similar to the activation enthalpy. While the deoxy form is characterized by an activation energy and enthalpy that is more than twice the corresponding value in the oxy form. The activation entropies show highly significant differences being ?128 e.u. and 136 e.u. at 25°C for the oxy and deoxy forms, respectively.     

A spectroscopic and electrochemical study of the interaction between copper (II) glycylglycyl-L-histidine-N-methylamide and iron (III) tetra-p-sulfophenylporphine

A binuclear complex has been produced by the reaction of an iron porphyrin (sodium tetra-p-sulfophenylporphine iron (III)-FeTPPS) with a copper metallo-tripeptide (copper (II) glycylglycyl-L-histidine-N-methylamide-CuGGH) in aqueous solution. The system has been characterized by electron spin resonance (ESR) spectroscopy, optical absorption spectroscopy, and electrochemical methods. Room-temperature ESR spectra of the copper complex and low-temperature ESR spectra of the iron porphine provide evidence for the formation of a binuclear complex. These findings are supported by absorption spectroscopy and electrochemical studies, and lead to a value of ca. 2 X 10(-3) M-1 (at room temperature) for the equilibrium constant for complex formation. The relevance of this system to the enzymic active site of mammalian cytochrome c oxidase is discussed.     

Circular dichroism (CD) studies on yeast enolase: activation by divalent cations

The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes. This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure. There was no effect of magnesium ion on the far ultraviolet spectrum. Evidently magnesium ion has no effect on the secondary structure. Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity. The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions. Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes. The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups. Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme. The "catalytic" and "inhibitory" sites did not appear to be very CD active.     

Isolation of a low molecular weight zinc binding ligand from human milk

A low molecular weight zinc binding compound from human milk has been purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Evidence is provided that this compound is citrate. A higher amount of citrate-bound zinc was found in human milk than in cow's milk. It is suggested that the therapeutic value of human milk for patients with the genetic disorder of zinc metabolism acrodermatitis enteropathica (AE) derives from a greater content of bioavailable zinc citrate in human than in cow's milk.     

Some studies on the metabolism of labelled molybdenum compounds in cattle

An initial experiment showed that [99Mo]di- and trithiomolybdates could be detected in bovine plasma after the introduction of [99Mo]molybdate into the rumen. It was felt that this justified the use of [99Mo]trithiomolybdate for the subsequent studies made of plasma thiomolybdate metabolism in vivo in cattle. Rapid intravenous injection of [99Mo]trithiomolybdate into cattle showed that doses of 50 mg Mo were subject to extensive hydrolysis over the first few minutes postinjection, but at lower dose rates this was reduced so that tracer doses (less than 1.5 mg Mo) were relatively stable. The plasma metabolism was unaffected by copper status within the limits of the experiments (that is, liver copper levels down to 9 mg/kg d.m.) The disappearance of [99Mo] and [35S]trithiomolybdate (1 mg Mo) from plasma was delayed for up to 10 hr by the immediate preinjection of copper, although no chemical modification of the thiomolybdate appeared to occur.     

Spectroscopic studies on the copper(II)—Inosine system

Interactions of inosine derivatives with copper(II) were studied in the pH range 1.4–13 in 50% H₂O-50% DMSO solution. The distinct pH dependence of the optical spectra observed in copper(II)-inosine complexes are correlated to their respective EPR changes as a function of pH. It was concluded that a simple 1:1 complex of copper(II)-inosine is formed in the pH range 1.4–5.0 and bis complexes are present in the pH 5.0–6.2 region solutions of inosine and Cu(II). From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex dominates. At pH 7.8–9.2 an insoluble, oxybridged species is formed in addition to the soluble paramagnetic Cu(NI)₄ complex. Starting from pH 9.1 the N-polymeric complex is formed which is stable up to pH 12.5, and above pH 12.5 the only species is the Cu(ribose)₂ complex.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

Solubility of calcium and zinc in model solutions based on bovine and human milks

The solubility of calcium, magnesium, and zinc in model solutions based on the low molecular weight components of bovine and human milks was examined over a pH range similar to that found in the human digestive system. Zinc was removed from solution in all models as calcium phosphates precipitated. The pH at which precipitable calcium phosphates formed was altered by the concentration of inorganic phosphate. All calcium and zinc in a model based on human milk remain in solution up to pH 6.5 while in a model based on bovine milk they were in solution up to pH 5. The use of simple model solutions may provide information useful for understanding the different bioavailabilities of minerals from skimmed bovine and human milks.     

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.     

Frequency and intensity of chick distress calls: Effects on maternal foodcalling in chickens

The food call of broody domestic hens was used to measure maternal response to four frequency components found in chick distress calls (2,3,4 and 5 kHz) and to variations in distress call intensity (0—86 dB). Foodcalling increased significantly with frequency of the pure-tone test pulse; response to a taped distress call occurred between 40 dB and 86 dB intensity with a maximum at 60–65 dB. The results suggest that the mother uses the higher frequency components in recognizing the distress call, but responds maximally within a specific intensity range. The selective advantage of such behaviour is discussed.     

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.     

Deep hypothermia and circulatory arrest: effect on cerebrospinal fluid electrolytes in newborn lambs.

Cerebrospinal fluid (CSF) Na, K, and acid-base changes were studied in 13 new-born lambs anesthetized with α-chloralose (60 mg/kg) or diethylether during 90 min of normothermic (37 °C) or hypothermic (20 °C) circulatory arrest. CSF K concentration increased linearly from 3.1 to 23.2 meq/liter during 90 min of normothermic circulatory arrest. During hypothermic circulatory arrest, animals anesthetized with α-chloralose exhibited an exponential increase in CSF K concentration from 3.1 to 13.6 meq/liter and animals anesthetized with diethylether had an exponential increase in CSF K concentration from 3.3 to 12.7 meq/liter. The rate of increase in CSF K concentration in hypothermic and normothermic animals between 60 and 90 min of circulatory arrest was the same. CSF Na concentration decreased slightly in both hypothermic and normothermic animals, with a greater decrease in the normothermic group.Although CSF pH and bicarbonate were significantly decreased during normothermia as well as hypothermia, both CSF pH and bicarbonate showed greater decreases during normothermia. Mean pH values after 90 min of circulatory arrest were 6.34, 6.87, and 6.77, respectively, in the normothermic, α-chloralose-hypothermic, and diethylether-hypothermic groups; corresponding values for bicarbonate were 7.7, 13.8, and 12.2 meq/liter.CSF pCO₂ increased linearly from 40.2 to 190.0 Torr during 90 min of normothermic circulatory arrest, from 28.6 to 92 Torr in the ether-hypothermic group, and from 28 to 81 Torr in the α-chloralose-hypothermic group.     

Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.     

Effect of acute stress on the absorption and distribution of zinc and on Zn-metallothionein production in the liver of the chick.

A study has been made of the effects of chloroform inhalation, Escherichia coli endotoxin injection and hydrocortisone injection on the absorption of a single intragastric dose of 65Zn by the chick. Injection of hydrocortisone increased the absorption of the 65Zn by 30-55% in both Zn-deficient and Zn-supplemented chicks. The influence of chloroform and endotoxin was less consistent; the former treatment only increased 65Zn absorption and endotoxin was less consistent; the former treatment only increased 65Zn absorption in Zn-supplemented chicks fed ad libitum whereas endotoxin only increased that in Zn-supplemented chicks on a restricted food intake. Injection of endotoxin increased the hepatic uptake of the absorbed 65Zn in both Zn-deficient and Zn-supplemented chicks, whereas hydrocortisone had a similar effect in the Zn-supplemented birds only. Chloroform inhalation increased hepatic 65Zn uptake in Zn-deficient chicks only. The increase in hepatic Zn concentrations in the stressed chicks was mainly associated with a protein in the cytosol identified as metallothionein. Both endotoxin and hydrocortisone decreased total plasma Zn concentrations in Zn-supplemented and Zn-deficient chicks; chloroform decreased plasma 65Zn content only.     

The thermodynamics of lanthanide ion binding to adriamycin

We have examined the thermodynamics of lanthanide ion binding to adriamycin by monitoring the effects of variations in temperature on the dissociation constants of various lanthanide ion complexes of the drug. These constants were obtained by analyzing the extent of quenching of the fluorecence of adriamycin in the presence of lanthanide ions in terms of an equilibrium binding process. Our binding model included the following features, all of which are supported by evidence derived from previous published reports, vide infra. The lanthanides form 1:1 complexes with adriamycin. The binding is dependent on the pH of the solution, indicating that only the nonprotonated amine form of the drug participates in lanthanide ion binding. And finally the drug self-associates in solution to for a dimeric species. Our present results indicate that the binding process is almost completely independent of temperature, indicating that the enthalpy of complex formation is extremely small. The entropy terms are consistent with the formation of a complex in which the adriamycin acts as a bidentate ligand. Our results suggest that the lanthanide complexes are isostructural, at least as far as the adriamycin is concerned, throughout the lanthanide series.     

The copper-enzyme dopamine beta-monooxygenase: studies on the ability of several metals to inhibit the enzyme activity and to replace the copper

The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme.