The conformation and orientation of copper(II)-bleomycin intercalated with DNA

EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(II)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(II)-bleomycin-DNA containing one Cu(II)-bleomycin to 200 basepairs. EPR measurements were made at room temperature and 90 K for different orientations of the external magnetic field with respect to the helical axes of the fibers. The g-values ($\text{g}{}_{\mathrm{}}\text{=2.21}$, g_⊥ =2.04) and the hyperfine constant ($\text{A}{}_{\mathrm{}}\text{=175}\text{G}$) are consistent with values expected for Cu(II) chelated to a square planar array of ligands. In the oriented fibers, the square planar arrays do not all have the same orientations with respect to the fiber axes. At room temperature the chelated ions have rotational freedom in which the normal to the planar array has almost complete freedom of rotation about axes perpendicular to the DNA fiber axes. The normal maintains an angle of 75° with respect to the axis, in the plane of the basepair, about which it rotates. Nine superhyperfine peaks on the high field side of the EPR spectrum were partially resolved. The number and splitting (12 G) of these superhyperfine peaks indicate that four nitrogen atoms are chelated to Cu(II) in a square planar array. These data on Cu(II)-bleomycin bound to DNA give information on the orientation of the metal-containing portion of bleomycin which lies outside the double helix.     

14N-ENDOR evidence for imidazole coordination in copper proteins

¹⁴N-ENDOR studies of simple nitrogen-coordinated copper(II) complexes in frozen aqueous solutions show that the nitrogen hyperfine constants, A_″ and A_⊥, of imidazole are much more isotropic ($\text{R =}\text{A}{}_{\mathrm{″}}\text{A}{}_{\mathrm{\perp }}\text{= 1.05}$) than those of the other biologically-related ligand nitrogens. From this result, combined with ¹⁴N-ENDOR results of some copper proteins containing imidazoles as ligands, it is concluded that R < 1.10 for nitrogen hyperfine constants can be employed as an empirical criterion for demonstration of the existence of imidazole coordination in copper proteins.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Ubisemiquinone radicals from the cytochrome b-c1 complex of the mitochondrial electron transport chain--demonstration of QP-S radical formation

Stable ubisemiquinone radical(s) in the cytochrome $\text{b}\text{?}\text{c}{}_1$-II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q₂. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (～9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved $\text{g}$-anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome $\text{b}\text{-}\text{c}{}_1$. This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome $\text{c}$ reductase from succinate dehydrogenase and the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

Stimulation by manganese and other divalent cations of the electron donation reactions of Photosystem II

Using inside-out thylakoid membranes, it has been shown that the oxidation of water and associated reduction of dichlorophenol indophenol is partially inhibited by low concentrations of cation chelators. This inhibition correlates with a removal of two manganese ions per Photosystem II reaction centre. The chelator-induced inhibition was completely reversed by the addition of low levels of Mn²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 20 μ}\text{M}$) and higher levels of Mg²⁺ and Ca²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{mM}$). Other cations were not effective, indicating that the ability to overcome the inhibition did not involve a general electrostatic screening process. The degree of inhibition by chelators was greater at lower light intensities and after treatment with glutaraldehyde. In the presence of glutaraldehyde the stimulatory effect of Mn²⁺ was lost, while pretreatment with Mn²⁺ prevented the glutaraldehyde effect. These results are discussed in terms of conformational changes of the electron donation chains involving cation- (preferentially Mn-) dependent coupling between the oxygen evolving and reaction-centre complexes of Photosystem II.     

Electronic interactions between iron and the bound semiquinones in bacterial photosynthesis. EPR spectroscopy of oriented cells of Rhodopseudomonas viridis

Electron paramagnetic resonance (EPR) spectroscopy of the iron-semiquinone complex in photosynthetic bacterial cells and chromatophores of Rhodopseudomonas viridis is reported. Magnetic fields are used to orient the prolate ellipsoidal-shaped cells which possess a highly ordered internal structure, consisting of concentric, nearly cylindrical membranes. The field-oriented suspension of cells exhibits a highly dichroic EPR signal for the iron-semiquinone complex, showing that the iron possesses a low-symmetry ligand field and exists in a preferred orientation within the native reaction-center membrane complex. The EPR spectrum is analyzed utilizing a spin hamiltonian formalism to extract physical information describing the electronic structure of the iron and the nature of its interaction with the semiquinones. Exact numerical solutions and analytical expressions for the transition frequencies and intensities derived from a perturbation theory expansion are presented, and a computer-simulated spectrum is given. It has been found that, for a model which assumes no preferred orientation within the plane of the membranes, the orientation of the Fe²⁺ ligand axis of largest zero-field splitting (Z, the principal magnetic axis) is titled 64±6° from the membrane normal. The ligand field for Fe²⁺ has low symmetry, with zero-field splitting parameters of |D₁|=7.0±1.3 cm^?1 and |E₁|=1.7±0.5 cm^?1 and $\text{|}\text{E}{}_1\text{D}{}_1\text{|=0.26}$ for the redox state Q₁^?Fe²⁺Q^2?. The rhombic character of the ligand field is increased in the redox state Q₁Fe²⁺Q^?₂, where $\text{0.33>|}\text{E}{}_2\text{D}{}_2\text{|>0.26}$. This indicates that the redox state of the quinones can influence the ligand field symmetry and splitting of the Fe²⁺. There exists an electron-spin exchange interaction between Fe²⁺ and Q^?₁ and Q^?₂, having magnitudes |J₁|=0.12±0.03 cm^?1 and $\text{|J}{}_2\text{|?0.06}\text{cm}{}^{\mathrm{?1}}$, respectively. Such weak interactions indicate that a proper electronic picture of the complex is as a pair of immobilized semiquinone radicals having very little orbital overlap (probably fostered by superexchange) with the Fe²⁺ orbitals. The exchange interaction is analyzed by comparison with model systems of paramagnetic metals and free radicals to indicate an absence of direct coordination between Fe²⁺ and Q^?₁ and Q^?₂. Selective line-broadening of some of the EPR transitions, involving Q^? coupling to the magnetic sublevels of the Fe²⁺ ground state, is interpreted as arising from an electron-electron dipolar interaction. Analysis of this line-broadening indicates a distance of 6.2–7.8 ? between Fe²⁺ and Q^?₁, thus placing Q₁ outside the immediate coordination shell of Fe²⁺.     

Electron paramagnetic resonance study of the oxidation of N-methyl-substituted aromatic amines catalyzed by hemeproteins

The oxidation of N-mono- and dimethyl-substituted toluidines and aniline by H₂O₂, catalyzed by horseradish peroxidase or metmyoglobin, produces organic free radicals, detectable by electron paramagnetic resonance spectroscopy at room temperature. The radical cation of N,N-dimethyl-p-toluidine was conclusively identified, but the other resolved EPR signals were assigned to radical cations of radical dimerization products, e.g., N,N,N′,N′-tetramethylbenzidine formed from N,N-dimethylaniline. The N-demethylase activities of metmyoglobin were found to be uniformly smaller than those of horseradish peroxidase, consistent with the much faster reaction of the latter hemeprotein with H₂O₂. Detection of the monomeric radical cation of N,N-demethyl-p-toluidine correlated with the largest rate of N-demethylation among this class of compounds. These findings emphasize the importance of radical stability (provided, for example, by the para methyl substituent) on subsequent competing reactions of the radical cation of the N-methyl substrate, i.e., one-electron oxidation leading to formaldehyde release or radical dimerization, which becomes more probable for the less stable radical intermediates. Attempts were made to correlate these results with data obtained for the $\text{O}{}_2\text{NADPH}\text{-supported}$ oxidation of these same substrates by liver microsomal cytochrome P-450. However, pronounced differences in physical state and kinetic properties of this heterogeneous, membrane-associated microsomal hemeprotein and the soluble “model” hemeprotein systems precluded firm conclusions concerning a radical mechanism of N-demethylation monooxygenase activities of microsomal fractions.     

The one-electron transfer redox potentials of free radicals. I. The oxygen/superoxide system

The method of determination of Redox potentials of radicals, using the pulse radiolysis technique, is outlined. The method is based on the determination of equilibrium constants of electron transfer reactions between the radicals and appropriate acceptors. The limitations of this technique are discussed.The redox potentials of several quinones-semiquinones are calculated, as well as the standard redox potential of the peroxy radical. and the redox oxidation properties of the peroxy radical in various systems and pH are discussed. The value determined for the redox potentials of $\text{O}{}_2\text{O}{}_2{}^{\mathrm{?}}$ is higher by more than 0.2 V than earlier estimates, which has important implications on the possible role of O₂^? in biological processes of O₂ fixation.     

Flash-induced carotenoid radical cation formation in Photosystem II

Light excitation of chloroplasts at low temperature produces absorption changes (ΔA) with a large positive peak at 990 nm and a bleaching around 480 nm. ΔA at 990 nm rises with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.6}\text{ms}$ at 20–77 K and remains largely stable. This signal is not observed when Photosystem II (PS II) photochemistry is blocked by reduction of the primary plastoquinone. It is observed also in purified PS II particles, in which case it could be shown that during a sequence of short flashes, the absorption at 990 nm rises in parallel with plastoquinone reduction measured at 320 nm. In chloroplasts the light-induced 990-nm ΔA at 77 K is increased under oxidizing conditions (addition of ferricyanide) and upon addition of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT2p). At 21°C, flash excitation of chloroplasts or of PS II particles induces only a very small ΔA at 990 nm, even when this is measured with a 100-ns time resolution or when the material is preilluminated. In both materials, however, a large flash-induced ΔA takes place when various lipophilic anions are added. After a flash the signal rises in less than 100 μs and its decay varies with experimental conditions; the decay is strongly accelerated by benzidine. The difference spectrum measured in PS II particles includes a broad peak around 990 nm and a bleaching around 490 nm. These absorption changes are attributed to a carotenoid radical cation formed at the PS II reaction center. It is estimated that in the presence of lipophilic anions at room temperature, one cation can be formed by a single flash in 80% of the reaction centers. At cryogenic temperature approx. 8% of the PS II reaction centers can oxidize a carotenoid after a single flash.     

A flash photolysis ESR study of Photosystem II Signal IIvf, the physiological donor to P-680+

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II ($\text{g ≈ 2.0045}$, $\text{ΔH}{}_{\mathrm{<mtext>pp</mtext>}}\text{≈ 19}\text{G}$). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with “red” illumination ($\text{λ < 680}\text{nm}$ and occurs stoichiometrically in a 1 : 1 ratio with $\text{P-700}{}^{\mathrm{+}}$. The Photosystem II transient is formed in less than 100 μs and decays via first-order kinetics with a halftime of 400–900 μs. Additionally, the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for radical decay is temperature independent between 20 and 4 °C; however, below 4 °C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal · mol^?1. Inhibition of electron transport through Photosystem II by $\text{o-}\text{phenanthroline}$, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ (1–5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this “quinoidal” type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, $\text{P-680}{}^{\mathrm{+}}$.     

The effect of redox potential on the kinetics of fluorescence induction in Photosystem II particles from Phormidium laminosum. Sigmoidicity,energy transfer and the slow phase

Fluorescence induction curves in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited Photosystem (PS) II particles isolated from the blue-green alga Phormidium laminosum have been analysed as a function of redox potential. Redox titration of the initial fluorescence indicated a single component with E_m,7.5 = +30 mV (n = 1) (Bowes, J., Horton, P. and Bendall, D.S. (1981) FEBS Lett. 135, 261–264). Despite this simplified electron acceptor system and the small number of chlorophylls per reaction centre, a sigmoidal induction curve was nevertheless seen. Sigmoidicity decreased as Q was reduced potentiometrically prior to induction such that the induction was exponential when the ratio $\text{F}{}_{\mathrm{<mtext>i</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}\text{= 0.64}$. These particles also showed a slow (β) phase of induction which titrated with an E_m value slightly more positive than that of the major quencher. It is concluded that the sigmoidal shape of the fluorescence induction curve observed in Phormidium PS II particles is not a consequence of a requirement for two photons to close the PS II reaction centre, but is generated as a result of energy transfer between photosynthetic units comprising one reaction centre per approx. 50 chlorophylls. Also, the existence of PS II heterogeneity (PS II_α, PS II_β centres) does not require a structurally differentiated chloroplast, but may only indicate the extent of aggregation of PS II centres.     

The reduction of the oxygen-evolving system in chloroplasts by thylakoid components

Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y₄) is lower compared to Y₃ than at lower flash frequency. At 4 Hz, the calculated S₀ concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent $\text{S}{}_0\text{(}\text{S}{}_0\text{+}\text{S}{}_1\text{)}$ ratio increases to about 0.2. This is explained by a relatively fast donation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.0–1.5}\text{s}$) of one electron by an electron donor to S₂ and S₃ in 15–25% of the Photosystem II reaction chains. The one-electron donor to S₂ and S₃ appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal II_s. (ii) The probability for the fast one-electron donation to S₂ and S₃ has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S₂ decay becomes 10-fold faster ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5–6}\text{s}$) in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S₂ decay was extremely slow. The S₃ decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S₂ is reduced mainly by Q^?_A, whereas S₃ is not. (iii) In the absence of CO₂/HCO^?_A and in the presence of formate, the fast one-electron donation to S₂ and S₃ does not occur. Addition of HCO^?₃ restores the fast decay of part of S₂ and S₃ to almost the same extent as in control thylakoids. The slow phase of S₂ and S₃ decay is not influenced significantly by CO₂/HCO^?₃. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q^?_A oxidation without interference of Q_B, were 2.3-fold slower in the absence of CO₂/HCO^?₃ than in its presence. (iv) An almost 3-fold decrease in decay rate of S₂ is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q^?_A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q^?_B is responsible for this shift of equilibrium Q^?_A concentration, these data would suggest that the pK_a value for Q^?_B protonation is somewhat higher than 7.6, assuming that the protonated form of Q^?_B cannot reduce Q_A.     

Photosystem I electron transport and phosphorylation supported by electron donation to the plastoquinone region

In chloroplasts, tetramethyl-p-hydroquinone supports high rates of phosphorylation-coupled, noncyclic electron flow through Photosystem I to methylviologen. The reaction is totally sensitive to dibromothymoquinone, indicating an electron donation to the plastoquinone region of the photosynthetic chain. The uncoupled electron flow rate exceeds 1000 μequivalents per hour per mg chlorophyll. The phosphorylation efficiency ($\text{P}\text{e}{}_2$) at the optimal pH of 8 is 0.6–0.65. Presumably this ratio represents the efficiency of energy coupling in the electron transfer step plastoquinone → cytochrome f.     

Chemical and enzymatic oxidation of alkylated benzo[a]pyrenes

The chemical and enzymatic oxidations of 6-, 7- and 10-methylbenzo[a]pyrenes, 6,10- and 7,10-dimethylbenzo[a]pyrenes and benzo[a]pyrene (BP) itself have been investigated to study the effects of alkyl substitution on the pathways of oxidation. The chemical oxidizing systems employed were Fenton's reagent ($\text{FeSO}{}_4\text{H}{}_2\text{O}{}_2$); trifluoroacetic acid-hydrogen peroxide (TFA/H₂O₂); thallium tristrifluoracetate in trifluoroacetic acid (TTFA/TFA) and H₂SO₄. The enzymatic systems were horseradish peroxidase (HRP/H₂O₂) and rat liver microsomes. The oxidations were investigated by electron paramagnetic resonance (EPR) spectroscopy to detect radical intermediates and by high performance liquid chromatography (HPLC) to separate the products. All the compounds studied produced radicals, identified as cationic species, in both H₂SO₄ and TTFA/TFA. In addition the 7-methyl-, 10-methyl- and 7,10-dimethyl-BP's produced 6-oxy radicals in some or all of the remaining oxidizing systems. Both chemically and enzymatically these same three compounds were observed to produce quinones as stable products. Microsomal oxidations gave the broadest range of products exhibiting HPLC peaks in the diol, quinone and phenol regions of the chromatograms, however, there were considerable differences between products from the individual derivatives and those from the parent molecule. 6-Methyl and 6,10-dimethyl-BP's showed no evidence of oxy radical intermediates or quinones under any set of conditions, the 6-substituent effectively blocking this oxidation pathway. The observations are consistent with the expected effects of alkyl substituents at particular positions and indicate that studies such as this one are potentially useful in better understanding oxidation and possible activation pathways of polycyclic aromatic hydrocarbons.     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Hemolysis caused by polyoxyethylene-derived surfactants evidence for peroxide participation

The hemolytic properties of nonionic surfactants of the series $\text{CH}{}_3\text{(}\text{CH}{}_2\text{)}{}_{\mathrm{15–17}}\text{-}\text{O}\text{-(}\text{CH}{}_2\text{CH}{}_2\text{O}\text{)}{}_{\mathrm{n}}\text{CH}{}_2\text{CH}{}_2\text{OH}$ were investigated and compared to those of saponins, sapogenins and H₂O₂. Antioxidants and anaerobic conditions were shown to inhibit the hemolysis, while glycyrrhizin was found to enhance it. Similar effects were obtained for H₂O₂ hemolysis, but not for saponin and sapogenin hemolysis. It is proposed that peroxides and free radicals are mainly responsible for the polyoxyethylene derived surfactants induced hemolysis.