Light-induced alkalization of the chloroplast stroma in vivo as estimated from the CO2 capacity of intact sunflower leaves

Rapid CO₂ gas exchange by Helianthus leaves was analysed kinetically using a computer model which distinguished different components of the gas exchange by different time constants. A rapid phase of CO₂ uptake was ascribed to the solubilization of CO₂ in all leaf compartments and to the conversion of the dissolved CO₂ to HCO₃⁻ in the chloroplast stroma which contains carbonic anhydrase. From stromal $\text{HCO}{}_3{}^{\mathrm{-}}\text{CO}{}_2$ ratios the stroma pH of darkened leaves was estimated to be close to 7.5. Occasionally, values as high as 8 or as low as 7 were also obtained. If fast HCO₃⁻ formation also occurs in the cytosol, pH values may be lower by about 0.3 pH units than those calculated under the assumption that carbonic anhydrase is localized in chloroplasts only. Illumination with a light intensity close to saturation of photosynthesis caused an increase in CO₂ solubilization which indicated the alkalization of the chloroplast stroma by about 0.6 pH units. This is an underestimation, if the pH of cytosol decreases in the light liberating CO₂ by the action of carbon anhydrase. An alkalization of the stroma by 0.6 pH units indicates the export of about 450 nmol H⁺/mg chlorophyll from the stroma. This forms the basis of a large transthylakoid pH gradient which drives light-dependent ATP synthesis. A pH gradient between stroma and cytosol is capable of supporting secondary gradients between these compartments in the light, such as a gradient in the $\text{ATP}\text{ADP}$ ratio. On darkening, the stroma alkalization was reversed. The rate of stroma acidification was much higher in the presence of CO₂ than in its absence.     

Bicarbonate ion-ATPase in rat liver cell fractions

The distribution of $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity. Approximately 85 % of the total $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity of the post 6000 x g · min supernatant was recovered in the mitochondrialfraction. The properties of this mitochondrial $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ were not distinguishable from those of the various microsomal $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ previously described by other investigators.     

Correlation between insulin action upon glycolysis and change in intracellular pH.

In the presence of $\text{CO}{}_2\text{HCO}{}_3{}^{\mathrm{?}}$, insulin both increases intracellular pH and stimulates glycolysis in frog skeletal muscle. When the action of insulin upon intracellular pH is blocked, either by amiloride or by decreasing extracellular sodium fifteen fold, the effect of the hormone upon glycolysis is also blocked.     

Carbonic anhydrase catalyzed oxygen-18 exchange between bicarbonate and water

Oxygen-18 exchange techniques were applied to the dehydration of bicarbonate catalyzed by human carbonic anhydrase C. The rates of depletion of oxygen-18 from labeled bicarbonate were measured for both the catalyzed and uncatalyzed reactions at pH 9.4 and 25 °C. The equilibrium dissociation constant of the enzyme-substrate complex K is 0.321 ± 0.040 m and $\text{k}{}_{\mathrm{<mtext>enz</mtext>}}\text{=}\text{k}{}_2\text{K}{}_{\mathrm{m}}$ is (8.3 ± 1.9) × 10⁵m^?1 sec^?1 under these conditions. On the basis of these results it is demonstrated that the oxygen-18 exchange technique is capable of measuring K and k_enz for the carbonic anhydrase catalyzed dehydration of bicarbonate at a high pH range in which other kinetic techniques are not effective.It was also shown that the oxygen-18 exchange technique is an effective micromethod for the determination of carbonic anhydrase. Rates of isotopic depletion of labeled bicarbonate (in solutions of the enzyme) which fall outside the limits of error for the uncatalyzed rate of depletion demonstrate that this technique can detect concentrations of human carbonic anhydrase C as low as 5 × 10^?11m.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

Superoxide anion as a cofactor of dopamine-beta-hydrxylase.

Superoxide anion can serve a reducing agent for tyramine hydroxylation by dopamine-β-hydroxylase. Stable $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ solutions were obtained by dissolving KO₂ in dry dimethylsulfoxide and infused into buffered solutions of tyramine and dopamine-β-hydroxylase at constant rate. The reaction requires molecular oxygen, but differs from the ascorbate dependent hydroxylation in its alkaline pH optimum value (pH 7.5) and its low rate (9 nmol octopamine formed/min/mg of protein). In absence of tyramine $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ does not produce a stable reduced form of the enzyme.     

Mercaptide-anion as a trans-ligand in oxycytochrome P450

Oxygenation of heme-mercaptide as well as spectroscopic characteristics of the dioxygen complex formed have been studied. Absorption and magnetic circular dichroism spectra of the O₂ complex support the retention of mercaptide in the heme fifth position. A release of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ in the decomposition of the oxygenated complex and an independent formation of the latter from hemine-dimercaptide and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ together with electron paramagnetic resonance and Mössbauer data support the O₂ presence in the heme coordination sphere. The similarity of optical and magnetic circular dichroism spectra and the closeness of the ratio for oxy-heme-mercaptide and oxycytochrome P450 unequivocally confirm the presence of an axial cystein mercaptide ligand in oxycytochrome P450.     

Interaction of human fibrinogen with divalent metal chlorides

Precipitation of human fibrinogen in 0.15 m NaCl occurred at pH 7.4 (Tris-HCl buffer) when ZnCl₂, CuCl₂, NiCl₂, or CoCl₂ were added beyond their respective critical concentrations. The critical concentrations were about $\text{4 × 10}{}^{\mathrm{?5}}\text{m}\text{ZnCl}{}_2\text{, 6 × 10}{}^{\mathrm{?5}}\text{m}\text{CuCl}{}_2\text{, 3 × 10}{}^{\mathrm{?4}}\text{m}\text{NiCl}{}_2\text{and}\text{1 × 10}{}^{\mathrm{?3}}\text{m}\text{CoCl}{}_2$. At pH 5.8 2-(N-morpholino)-ethane sulphonic acid buffer, the critical concentrations were found only for CuCl₂ and ZnCl₂, and were about $\text{3 × 10}{}^{\mathrm{?5}}\text{and}\text{3 × 10}{}^{\mathrm{?4}}\text{m}$, respectively. CaCl₂ and MgCl₂ were not effective up to $\text{1 × 10}{}^{\mathrm{?2}}\text{and}\text{2 × 10}{}^{\mathrm{?2}}\text{m}$ at pH 7.4 and 5.8, respectively. At pH 7.4, precipitation was better in 0.015 m NaCl than in 0.15 m NaCl for both CuCl₂ and ZnCl₂. Little or no conformational change was indicated on binding Cu²⁺ ions. The fluorescence of tryptophan was quenched only by CuCl₂, while other metal ions (ZnCl₂, NiCl₂, CoCl₂ and CaCl₂) were ineffective as quenchers.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

Effect of reduced pH in absence of HCO3− on anomalous and normal potential responses in bullfrog antrum

Effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO₃^? but with 95% O₂/5% CO₂ in both cases. In both cases, changing from 4 to 40 mM K⁺ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO₃^?. Changing from 102 to 8 mM Na⁺ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO₃^? was insignificant but with 25 mM HCO₃^? the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K⁺ and Na⁺ were largely related to ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pump. Changes in nutrient Cl^? from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pump and Na⁺/Cl^? symport. Removal of HCO₃^? and concurrent reduction of pH modify resistance of these pathways.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

The structure of the fluorophosphate-pyruvate kinase complex investigated by 31P relaxation rate studies

The interaction of fluorophosphate with muscle pyruvate kinase was investigated by ³¹P nuclear relaxation rate measurements. The fluorophosphate samples were highly purified and were first monitored by ¹⁹F and ³¹P relaxation rate measurements in the formation of the binary FPO₃-Mn complex. The results of the binary complex demonstrated that FPO₃^2? binds in the first coordination sphere of Mn²⁺ via the oxygen atoms but not via the fluorine. The enzyme experiments were designed under conditions where a significant fraction of the ligand is in the ternary enzyme-Mn-FPO₃ complex. These studies demonstrate that the ³¹P relaxation rate of bound FPO₃ ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>m</mtext>}}\text{= 1.58 ± 0.05 × 10}{}^5\text{s}{}^{\mathrm{?1}}$) is consistent with the binding of this ligand in the first coordination sphere of enzyme-bound Mn²⁺ with an elongated Mn-O-P distance ($\text{r}{}_{\mathrm{<mtext>Mn-P</mtext>}}\text{= 3.3 ± 0.2}\text{A}\text{?}$). Such a structure is demonstrated in the ternary enzyme-Mn-FPO₃ complex, in the complex containing HCO₃^?, and in the complex also containing HCO₃^? and ADP. The data further substantiate the binding of phosphoenolpyruvate analogs in the first coordination sphere of pyruvate kinase-bound Mn²⁺.     

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K⁺ in the presence of valinomycin, and cation influx was coupled to an efflux of H⁺ using the protonophore $\text{3-tert-}\text{butyl-5,2′-dichloro-4′-nitrosalicilylanilide}$. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be $\text{SCN}{}^{\mathrm{?}}\text{> I}{}^{\mathrm{?}}\text{> CH}{}_3\text{COO}{}^{\mathrm{?}}\text{> Cl}{}^{\mathrm{?}}\text{≈ HCO}{}^{\mathrm{?}}{}_3\text{≈ P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2?}}$ and that to cations $\text{Cs}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> H}{}^{\mathrm{+}}$. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.     

Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2−• generating oxidase from human neutrophils

The resolved flavoprotein and cytochrome b₅₅₉ components of the NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b₅₅₉, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b₅₅₉, depleted of flavoprotein, demonstrated no measureable NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b₅₅₉ was rapidly oxidized by oxygen, as was the cytochrome b₅₅₉ in the intact oxidase.     

Reactivity of chemically generated superoxide radical anion with peroxides as determined by competition kinetics

A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. High pressure liquid chromatography is used as an assay system for $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene.