Redshift of the purple membrane absorption band and the deprotonation of tyrosine residues at high pH: Origin of the parallel photocycles of trans-bacteriorhodopsin

At high pH (> 8) the 570 nm absorption band of all-trans bacteriorhodopsin (bR) in purple membrane undergoes a small (1.5 nm) shift to longer wavelengths, which causes a maximal increase in absorption at 615 nm. The pK of the shift is 9.0 in the presence of 167 mM KCl, and its intrinsic pK is ~8.3. The red shift of the trans-bR absorption spectrum correlates with the appearance of the fast component in the light-induced L to M transition, and absorption increases at 238 and 297 nm which are apparently caused by the deprotonation of a tyrosine residue and red shift of the absorption of tryptophan residues. This suggests that the deprotonation of a tyrosine residue with an exceptionally low pK (pK_a ≈ 8.3) is responsible for the absorption shift of the chromophore band and fast M formation. The pH and salt dependent equilibrium between the two forms of bR, “neutral” and “alkaline,” bR ↔ bR_a, results in two parallel photocycles of trans-bR at high pH, differing in the rate of the L to M transition. In the pH range 10-11.8 deprotonation of two more tyrosine residues is observed with pK's ~ 10.3 and 11.3 (in 167 mM KCL). Two simple models discussing the role of the pH induced tyrosine deprotonation in the photocycle and proton pumping are presented.

It is suggested that the shifts of the absorption bands at high pH are due to the appearance of a negatively charged group inside the protein (tyrosinate) which causes electrochromic shifts of the chromophore and protein absorption bands due to the interaction with the dipole moments in the ground and excited states of bR (Stark effect). This effect gives evidence for a significant change in the dipole moment of the chromophore of bR upon excitation.

Under illumination alkaline bR forms, besides the usual photocycle intermediates, a long-lived species with absorption maximum at 500 nm (P500). P500 slowly converts into bR_a in the dark. Upon illumination P500 is transformed into an intermediate having an absorption maximum at 380 nm (P380). P380 can be reconverted to P500 by blue light illumination or by incubation in the dark.

     

Light-induced currents from oriented purple membrane: I. Correlation of the microsecond component (B2) with the L-M photocycle transition

When irradiated, purple membrane from Halobacterium halobium oriented in a polyacrylamide gel produces a photocurrent. The correlation of the microsecond component (B2) of the photocurrent with the L-M optical transition was studied. It was found that the lifetimes of B2 and the L-M transition are identical over the entire pH range from 2.4 to 11.0 when measured in high salt (>5 mM CaCl₂ or >40 mM KCl). Changing the temperature from 10 to 35°C, or replacing the H₂O with D₂O maintains this correlation. The amplitude of B2 and the L-M transition are also correlated over the pH range where both of them can be represented as a single exponential. At high pH (>8), three exponentials were required to fit both the optical and photocurrent signals. Two of them are the previously described fast and slow components of M formation, but a new intermediate with a very fast lifetime, 0.3 μs, was observed both in absorption (λ = 410 nm) and photocurrent measurements. The lifetimes of all three were found to be pH independent. This would exclude models for the L to M portion of the photocycle that explained its complex pH-dependent behavior as being due to a single pH-dependent rate constant. The area of B2, which is proportional to the number and the distance the charge moved during the transition, is almost constant from pH 5.0 to pH 8.0. It decreases to almost zero at pH 2.0 and pH 10.6 with pKs at 2.8 and 9.1. Because B2 is thought to normally reflect proton release from the membrane, this suggests at very low and high pH the photocycle does not pump protons. The pK at high pHs for the formation of the nonpumping photocycle is probably related to the formation of a new photocycle featuring the fast rising form of M.     

Gamma Study of pH, Nitrite, and Salt Inhibition of Aeromonas hydrophila

The gamma hypothesis states that there are no interactions between antimicrobial environmental factors. The time to growth of Aeromonas hydrophila challenged with pH, NaNO₂, and salt combinations at 30°C was investigated. Data were examined using a model based on the gamma hypothesis (the gamma model), which takes into account variance-stabilizing transformations and which gives biologically relevant parameters. At high concentrations of NaNO₂ and at pHs of >6.0, the antimicrobial action of the nitrite ion has a strong influence (MIC = 2,033 mg liter⁻¹), whereas at pHs of <6, nitrous acid is dominant (MIC = 1.5 mg liter⁻¹). This change is not due to a “synergy” between pH and the nitrite ion but is due to the shift in the equilibrium concentrations of nitrous acid and nitrite in solution caused by pH. In combination with salt, the parameters found for the action of Na nitrite were identical to those found when it was examined in isolation. Therefore, pH, NaNO₂, and salt act independently on the growth of A. hydrophila. By expanding the gamma model with a cardinal temperature model, the results of fitting the model of Palumbo et al. (J. Food Prot. 54:429-435, 1994) to randomly produced environmental conditions could be reproduced, suggesting that temperature also has an independent effect.     

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H₂O₂ became “available” during the oxidation of [1-¹⁴C]glycolate was sufficient to account for the breakdown of the intermediate [1-¹⁴C]glyoxylate to formate (C₁ unit) and ¹⁴CO₂. Under aerobic conditions formate production closely paralleled ¹⁴CO₂ release from [1-¹⁴C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H₂O₂ production was increased artificially by addition of an active preparation of fungal glucose oxidase.     

Molecular dynamics study of the 13-cis form (bR548) of bacteriorhodopsin and its photocycle.

The structure and the photocycle of bacteriorhodopsin (bR) containing 13-cis,15-syn retinal, so-called bR548, has been studied by means of molecular dynamics simulations performed on the complete protein. The simulated structure of bR548 was obtained through isomerization of in situ retinal around both its C13-C14 and its C15-N bond starting from the simulated structure of bR568 described previously, containing all-trans,15-anti retinal. After a 50-ps equilibration, the resulting structure of bR548 was examined by replacing retinal by analogues with modified beta-ionone rings and comparing with respective observations. The photocycle of bR548 was simulated by inducing a rapid 13-cis,15-anti-->all-trans,15-syn isomerization through a 1-ps application of a potential that destabilizes the 13-cis isomer. The simulation resulted in structures consistent with the J, K, and L intermediates observed in the photocycle of bR548. The results offer an explanation of why an unprotonated retinal Schiff base intermediate, i.e., an M state, is not formed in the bR548 photocycle. The Schiff base nitrogen after photoisomerization of bR548 points to the intracellular rather than to the extracellular site. The simulations suggest also that leakage from the bR548 to the bR568 cycle arises due to an initial 13-cis,15-anti-->all-trans,15-anti photoisomerization.     

Seasonality and Paleoecology of the Late Cretaceous Multi-Taxa Vertebrate Assemblage of “Lo Hueco” (Central Eastern Spain)

Isotopic studies of multi-taxa terrestrial vertebrate assemblages allow determination of paleoclimatic and paleoecological aspects on account of the different information supplied by each taxon. The late Campanian-early Maastrichtian “Lo Hueco” Fossil-Lagerstätte (central eastern Spain), located at a subtropical paleolatitude of ~31°N, constitutes an ideal setting to carry out this task due to its abundant and diverse vertebrate assemblage. Local δ¹⁸O_PO4 values estimated from δ¹⁸O_PO4 values of theropods, sauropods, crocodyliforms, and turtles are close to δ¹⁸O_H2O values observed at modern subtropical latitudes. Theropod δ¹⁸O_H2O values are lower than those shown by crocodyliforms and turtles, indicating that terrestrial endothermic taxa record δ¹⁸O_H2O values throughout the year, whereas semiaquatic ectothermic taxa δ¹⁸O_H2O values represent local meteoric waters over a shorter time period when conditions are favorable for bioapatite synthesis (warm season). Temperatures calculated by combining theropod, crocodyliform, and turtle δ¹⁸O_H2O values and gar δ¹⁸O_PO4 have enabled us to estimate seasonal variability as the difference between mean annual temperature (MAT, yielded by theropods) and temperature of the warmest months (TWMs, provided by crocodyliforms and turtles). ΔTWMs-MAT value does not point to a significantly different seasonal thermal variability when compared to modern coastal subtropical meteorological stations and Late Cretaceous rudists from eastern Tethys. Bioapatite and bulk organic matter δ¹³C values point to a C₃ environment in the “Lo Hueco” area. The estimated fractionation between sauropod enamel and diet is ~15‰. While waiting for paleoecological information yielded by the ongoing morphological study of the “Lo Hueco” crocodyliforms, δ¹³C and δ¹⁸O_CO3 results point to incorporation of food items with brackish influence, but preferential ingestion of freshwater. “Lo Hueco” turtles showed the lowest δ¹³C and δ¹⁸O_CO3 values of the vertebrate assemblage, likely indicating a diet based on a mixture of aquatic and terrestrial C₃ vegetation and/or invertebrates and ingestion of freshwater.     

New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy.

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.     

Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1^−/− skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K_1/2) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K_1/2 for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K_1/2 for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K_1/2 for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in “parallel” at alkaline pH. In the second “serial” model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.     

The pH-dependence of photochemical intermediates of O and P in bacteriorhodopsin by continuous light

The pH-dependence of the O and P intermediates in the photocycle of bacteriorhodopsin (bR) on the intensity and duration of the exciting flash was investigated for bR glycerol suspensions and bR gelatin films. Green and red laser flashes (532 and 670 nm) were utilized to generate a photoequilibrium state of bR and O at ambient temperature, and UV-vis spectroscopy was used to determine the photoconversion for the bR suspensions and films. The maximal concentration of the O intermediate was observed to be pH-dependent and the dependency was most pronounced at a slightly alkaline pH values. The photochemical conversion from the O to P intermediate was investigated for both bR suspensions and films. The P intermediate was only found in bR gelatin film. These results indicate that bR gelatin film may be an attractive candidate for the information storage based on P intermediate. It is possible, with red light, to create photoproducts which are thermally stable at ambient temperature and that can be photochemically erased.     

Utilization of Exogenous Inorganic Carbon Species in Photosynthesis by Chlorella pyrenoidosa

The nature of the inorganic carbon utilized during photosynthesis by Chlorella pyrenoidosa was investigated using three experimental techniques (open gas analysis system with “artificial leaf” or “aqueous” chambers and O₂ electrode system) to measure carbon assimilation. Photosynthesis was studied as a function of pH and CO₂ concentration. The CO₂ concentration was inadequate to meet the requirements of photosynthesis only when HCO₃⁻ was added at high pH. Under all other conditions, the low and constant K_m (CO₂), in contrast to the highly variable K_m (HCO₃⁻), suggested that CO₂ was the major species utilized.     

Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii

The “Phot” protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 μs with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.     

Forms of Dissolved Carbon Dioxide Required for Photosystem II Activity in Chloroplast Membranes

High concentrations of both bicarbonate and formate inhibit photosynthetic O₂ evolution at pH 8.0. At this pH, only 2.4% of the total dissolved carbon dioxide exists as CO₂. At pH 7.3, where 11% of the total dissolved carbon dioxide exists as CO₂, HCO₃⁻ no longer inhibits. While formate still inhibits O₂ evolution at pH 7.3, its effect can be partially overcome if CO₂ is also present. The rate of binding of added ¹⁴C-labeled inorganic carbon is nearly 10-fold more rapid when the internal pH of thylakoid membranes is at 6.0 than when it is at 7.8. These observations suggest that CO₂, not HCO₃⁻, is initially bound to the photosystem II reaction center and that the location of the binding site is on the inside thylakoid surface. However, additional data presented here suggest that, after binding, CO₂ is hydrated to HCO₃⁻ + H⁺ in a pH-dependent reaction. Two possible explanations of the “bicarbonate effect” are presented.     

The Effect of pH, O(2), and Temperature on the CO(2) Compensation Point of Isolated Asparagus Mesophyll Cells

The effect of pH, O₂ concentration, and temperature on the CO₂ compensation point (Г[CO₂]) of isolated Asparagus sprengeri Regel mesophyll cells has been determined in a closed, aqueous environment by a sensitive gas-chromatographic technique. Measured values range between 10 and 100 microliters per liter CO₂ depending upon experimental conditions. The Г(CO₂) increases with increasing temperature. The rate of increase is dependent upon the O₂ concentration and is more rapid at high (250-300 micromolar), than at low (30-60 micromolar), O₂ concentrations. The differential effect of temperature on Г(CO₂) is more pronounced at pH 6.2 than at pH 8.0, but this pH-dependence is not attributable to a direct, differential effect of pH on the relative rates of photosynthesis and photorespiration, as the O₂-sensitive component of Г(CO₂) remains constant over this range. The Г(CO₂) of Asparagus cells at 25°C decreases by 50 microliters per liter when the pH is raised from 6.2 to 8.0, regardless of the prevailing O₂ concentration. It is suggested that the pH-dependence of Г(CO₂) is related to the ability of the cell to take up CO₂ from the aqueous environment. The correlation between high HCO₃⁻ concentrations and low Г(CO₂) at alkaline pH indicates that extracellular HCO₃⁻ facilitates the uptake of CO₂, possibly by increasing the flux of inorganic carbon from the bulk medium to the cell surface. The strong O₂− and temperature-dependence of Г(CO₂) indicates that isolated Asparagus mesophyll cells lack an efficient means for concentrating intracellular CO₂ to a level sufficient to reduce or suppress photorespiration.     

Large Scale Global Structural Changes of the Purple Membrane during the Photocycle

Both the solution and the oriented film absorption and circular dichroic spectra of the bacteriorhodopsin (bR₅₆₈) and M₄₁₂ intermediate of the purple membrane photocycle were compared over the wavelength region 800-183 nm to assess structural changes during this photocycle. The main findings are (a) loss of the excitonic interaction among the chromophoric retinal transitions indicating disordering of the retinal orientations in the membrane and distortions of the membrane hexagonal crystal lattice, (b) structural change of the chromophoric retinal, (c) changes in the key interactions between the retinal and specific groups in the local environment of the apoprotein, (d) significant changes of the tertiary structure of the bR with negligible secondary structure involvement, and (e) a net tilting of the rodlike segments of the bR polypeptides away from the membrane normal. These findings are in accord with large scale global structural changes of the membrane during the photocycle and with structural metastability of the bR molecules. An important implication of these changes is the possibility of transmembrane retinal-regulated pulsating channels during the photocycle. The significance of this possibility in respect to models for the proton translocation function of this membrane is discussed.     

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of “electrical anisotropy” of retinal chromophore in the membranes. At about pH 7, there was no difference in the “electric anisotropy” between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the “electric anisotropy” are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of ~0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of ~0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 ±2 debye/bR.     

Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide

Chlorine dioxide (ClO₂) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO₂ multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct_99.99%) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min Ct_99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct_99.99% ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments, Ct_99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct_99.99% ranges and EFH model Ct_99.99% values demonstrated that FCV is more resistant to ClO₂ than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct_99.99% values are higher than Ct_99.99% values calculated from bench-scale experiments and from EFH model application.     

Protonation of a novel intermediate P is involved in the M → bR step of the bacteriorhodopsin photocycle

A novel intermediate (P) of the bacteriorhodopsin (bR) photocycle, appearing between M412 and bR is described. Like bR, intermediate P shows an absorption maximum at 560–570 nm. However, the extinction coefficient of P is somewhat lower than that of bR. Moreover, there are some differences in spectra of bR and P at wavelengths shorter than 450 nm. The P → bR transition correlates with the absorption of H⁺ from the water medium. The following conditions proved to be favourable for the detection of the new intermediate: a high salt concentration, low light intensity and low temperature (0.5°C). The P → bR transition is strongly decelerated by a small amount of Triton X-100. Illumination of P does not produce M412 before bR is formed. It is assumed that M412 converts to P when the Schiff base is protonated by a proton transferred from a protein protolytic group which participates in the inward H⁺-conductivity pathway. Reprotonation of this group results in the conversion of P to bR. No more than 1 H⁺ is transported per bR photocycle.     

Sporicidal properties of hydrogen peroxide against food spoilage organisms

The sporicidal properties of hydrogen peroxide were evaluated at concentrations of 10 to 41% and at temperatures of 24 to 76 C. The organisms tested and their relative resistance at 24 C to 25.8% H₂O₂ were: Bacillus subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus > Clostridium sp. putrefactive anaerobe 3679 > S. aureus, with „D” values of 7.3, 2, 1.8, 1.5, 0.8., and 0.2 min, respectively. Heat shocking spores prior to hydrogen peroxide treatment decreased their resistance. Wet spores were more resistant than dry spores when good mixing was achieved during hydrogen peroxide treatment. Inactivation curves followed first-order kinetics except for a lag period where the inactivation rate was very slow. Increasing the H₂O₂ concentration and the temperature reduced the lag period.     

Structural Effects of Cl and Other Anions on the Water Oxidizing Complex of Chloroplast Photosystem II

To further our understanding of the role of Cl⁻ and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl⁻ free media of pH ≈ 7 was enhanced by millimolar concentrations of the divalent anion SO₄²⁻ and also by divalent cations like Mg²⁺ and Ca²⁺; (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl⁻ = Br⁻ > NO₃⁻ > F⁻ > ClO₄⁻; (c) at molar concentrations, SO₄²⁻ stabilized the binding of the 23 kilodalton polypeptide, while Cl⁻ and Br⁻ became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0°C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl⁻, or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly, but is essential for the catalytic mechanism as well. Binding of, or charge screening by, divalent ions interferes with this process. At high salt concentrations, all these effects are overridden by “lyotropic” actions of the solutes that affect the integrity of the water oxidizing protein complex by stabilizing or disrupting critical hydrophobic domains.     

Defluorination of Aqueous Perfluorooctanesulfonate by Activated Persulfate Oxidation

Activated persulfate oxidation technologies based on sulfate radicals were first evaluated for defluorination of aqueous perfluorooctanesulfonate (PFOS). The influences of catalytic method, time, pH and K₂S₂O₈ amounts on PFOS defluorination were investigated. The intermediate products during PFOS defluorination were detected by using LC/MS/MS. The results showed that the S₂O₈ ²⁻ had weak effect on the defluorination of PFOS, while the PFOS was oxidatively defluorinated by sulfate radicals in water. The defluorination efficiency of PFOS under various treatment was followed the order: HT (hydrothermal)/K₂S₂O₈ > UV (ultraviolet)/K₂S₂O₈ > Fe²⁺/K₂S₂O₈ > US (ultrasound)/K₂S₂O₈. Low pH was favorable for the PFOS defluorination with sulfate radicals. Increase in the amount of S₂O₈ ²⁻ had positive effect on PFOS defluorination. However, further increase in amounts of S₂O₈ ²⁻ caused insignificant improvement in PFOS defluorination due to elimination of sulfate radicals under high concentration of S₂O₈ ²⁻. CF₃(CF₂)_nCOOH (n = 0–6) were detected as intermediates during PFOS defluorination. Sulfate radicals oxidation and hydrolysis were the main mechanisms involved in defluorination process of PFOS.