The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent K_m = 4 μM and saturation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is approx. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.     

Localized or delocalized protons in photophosphorylation? On the accessibility of the thylakoid lumen for ions and buffers

The deposition of protons inside thylakoids after flash excitation was measured photometrically with neutral red as pH indicator. In continuation of previous work (Junge, W., Ausländer, W., McGeer, A. and Runge, T. (1979) Biochim. Biophys. Acta 546, 121–141), we studied the influence of salts on neutral red binding and on the pK of the heterogeneous protonation-deprotonation of inside-bound neutral red as a function of salts. With freeze-thawed (cryoprotective dimethyl sulphoxide) or aged chloroplasts, we observed that the heterogeneous pK of inside-bound neutral red was salt dependent in a way which suggested that neutral red was bound close to the plane of negative fixed charges and that the adjacent inner aqueous phase could accommodate an extended ionic double layer. This, together with the known extremely rapid proton exchange between surface layer and adjacent bulk phase, led us to conclude that inside-deposited protons rapidly reached an aqueous inner bulk phase. This conclusion was corroborated by the observation that extremely hydrophilic buffers like phosphate quenched the transient internal acidification independent of whether proton deposition was due to water oxidation or to plastohydroquinone oxidation. Very different behaviour was observed for freshly prepared chloroplasts with broken outer envelope. Here, inside-bound neutral red was seemingly unaffected by salts and hydrophilic buffers failed to quench the internal acidification. The electrical conductivity and proton permeability of the thylakoid membrane, on the other hand, were as usual. We attributed the seeming inaccessibility of the internal phase to the failure to accommodate a sufficiently extended ionic cloud between the tightly appressed membranes. In such material we observed hindered lateral mobility of protons at the outer side of the thylakoid membrane. This was tentatively attributed to multiple binding-debinding at buffering groups. The consequences for the chemiosmotic theory are: There is one type of damaged chloroplast material, which is competent in photophosphorylation and where protons are deposited into an internal aqueous bulk phase in the orthodox sense. In more intact material, however, the internal space lacks the characteristic properties of an aqueous bulk phase and there is evidence for lateral diffusion limitation for protons. Here, the thermodynamics of photophosphorylation may be inadequately described by the proton-motive force between two aqueous phases which are each isopotential.     

Nonequilibration of membrane-associated protons with the internal aqueous space in dark-maintained chloroplast thylakoids

Isolated spinach thylakoids retain a slowly equilibrating pool of protons in the dark which are predominantly bound to buffering groups, probably amines, with low pKa values. We have measured the effects of permeant buffers, salts, sucrose, and uncouplers on the retention of the proton pool. Acetic anhydride, which reacts with neutral primary amine groups, was used to determine the protonation state of the amine buffering groups. It was previously shown by Bakeret al. that the extent of inhibition of photosystem II water-oxidizing capacity by acetic anhydride and the increase in derivatization by the anhydride are proportional to, and dependent on, the deprotonated state of the amine buffering pool. Therefore, acetic anhydride inhibition of water oxidation activity may be used as a measure of the protonation state of the amine buffering pool. By this method it is inferred that protons, in a metastable state, were retained by membranes suspended in high pH buffer for several hours in the dark. When both the internal and external aqueous phases were equilibrated with pH 8.8 buffer, the proton pool was released only upon addition of a protonophore. The osmotic strength of the suspension buffer affected uncoupler-induced proton release while ionic strength had little influence. The acetic anhydride-sensitive buffering group(s) of the water-oxidizing apparatus had an apparent pKa of 7.8. We conclude that an array of protein buffering groups reside either within the membrane matrix, or in proteins at the membrane surface, not in equilibrium with the bulk aqueous phases, and is responsible for the retention of the proton pool in dark maintained chloroplasts.     

Thylakoid volume, proton translocation and buffering capacity as measured with spin-label techniques

In model studies with lipid vesicles it is shown that the main population of the spin label Tempamine is bound to the membrane surfaces, the piperidine ring directed to the lipid phase. The signal of external label but not of label in the surface is broadened by chromium oxalate. Inner volumes of vesicles can be derived from the partially resolved polar part of the high-field line of Tempone or from the area of the Tempamine spectrum removed by making chromium oxalate enter the vesicles. If this membrane-associated population is corrected for, rotational correlation times for the label in the lumen can be obtained showing that hindrance of rotation is only by a factor of 3–5 instead of 10 as previously reported. In studies with thylakoids volumes of 1–3 μl/mg Chl were found with 0.3 M sorbitol as an osmoticum, 5 mM MgCl₂, 20 mM KCI, and 20 mM chromium oxalate. The internal buffering capacity and the magnitude of pH changes in the inner volume can be determined from flash-induced changes in the amine distribution. The buffering capacity is found to be 7–20 mM in buffer-permeable and approx. 100 mM in buffer-impermeable thylakoids, that is approx. 100 neq per mg Chl. The apparent H⁺/e⁻-value in impermeable preparations was found to be up to 0.7 and lower in permeabilized material. ΔpH per flash is 0.04–0.06 units. Possible sources of errors, particularly the presence of non-functional or non-thylakoid membranes, are discussed. Time-resolved signals are presented and several side effects and their suppression are discussed. The response time of the method is up to 2 ms, protons from the donor side of Photosystem II can be separated kinetically from those liberated by the intersystem chain. While transients with less than 2 ms and approx. 20 ms were found with ferricyanide as an electron acceptor in accordance with the results with neutral red, pronounced slow phases (t_1/2 is several hundred ms) were found without acceptors. Evidence is presented indicating that at least part of these responses do not originate from the thylakoid inner volume.     

The effect of low concentrations of uncouplers on the detectability of proton deposition in thylakoids. Evidence for subcompartmentation and preexisting pH differences in the dark

Flash-induced pH changes inside thylakoids were measured with neutral red as an indicator in the presence and absence of low concentrations of uncouplers. We found that both carrier-type and pore-forming uncouplers caused the rapidly rising phase of the neutral red signal, previously attributed to proton deposition by water oxidation, to disappear. Gramicidin was particularly efficient in this respect, requiring only one molecule of uncoupler per 10⁴ chlorophyll molecules to render the rapid proton deposition undectectable. This suggests that gramicidin did not act on each water-oxidizing enzyme individually, but rather at the level of the thylakoid membrane. In contrast to the effect on water-derived protons, the appearance of protons from plastoquinol was unaffected by gramicidin. Nor did gramicidin affect the rise of the neutral red signal due to proton deposition during two Photosystem I partial reactions with artificial donors. At the low gramicidin concentrations used, its effect on the neutral red signal could not be attributed to a general increase in proton permeability of the thylakoid membrane (acceleration of half decay from 9 to 0.8 s). The extent of alkalinization of the external medium during the first few hundred milliseconds following a light flash was unaltered by gramicidin, and we did not observe a kinetic correlation between the disappearance of the water proton and the decay of the transmembrane electric field. The last two findings suggest that the undetected protons had not crossed the thylakoid membrane, but instead were buffered away by some gramicidin-induced extra buffering power. pH titration of this extra buffering power revealed an apparent pK ranging between 7.2 and 7.7 and a stoichiometry of $\text{2}\text{H}{}^{\mathrm{+}}\text{site}$. The rapid phase of the neutral red signal regained 90% of its original amplitude after seven flashes were applied at 6.7 Hz repetition rate to a sample containing gramicidin. This suggests limits to the extra buffering power. One possible interpretation of our experiments is the following: Protons derived from water oxidation are initially deposited into extended and highly buffering special domains, and only escape into the common internal phase when the buffering capacity of the domains is saturated. As an alternative one may consider that the thylakoid lumen is partitioned into at least two domains, each dominated by different photosystems and with slow proton equilibration between them. Either view requires internal subcompartmentation. The consequences of such subcompartmentation for chloroplast bioenergetics are still obscure.     

Determination of buffering capacity of rat myocardium during ischemia

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.     

The slow rise of the flash-light-induced alkalization by Photosystem II of the suspending medium of thylakoids is reversibly related to thylakoid stacking

We studied the kinetics of flash-induced proton uptake at the reducing site of Photosystem II with Cresol red as indicator for pH transients in the suspending medium. The rise of the alkalization which was observed when Photosystem II was hidden in the stacked regions of the thylakoid membranes was much slower (100 ms) than the reduction of the bound quinones (less than 1 ms). We asked for the delay mechanism. We found that the rise of the alkalization became biphasic if thylakoids were unstacked. This was reversed upon restacking. The portion of the fast phase (half-rise time, 2.7 ms) increased, if the concentration of Mg²⁺ was lowered. After EDTA-treatment we observed solely fast proton uptake. Experiments with dark-adapted chloroplasts showed that the biphasicity was not attributable to the alternating transitions of the bound quinone acceptors through the semiquinone and the hydroquinone stages. The dependence of fast proton uptake on the degree of membrane stacking was on line with our previous proposal (Hong and Junge (1983) Biochim. Biophys. Acta 722, 197–205) that the propagation of a pH pulse in the narrow gaps between stacked membranes was slowed down by multiple reactions of protons with fixed buffering groups. This concept was corroborated by a theory which was given in the subsequent article (Junge, W. and Polle, A. (1986) Biochim. Biophys. Acta 848, 265–273).     

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers

(1) The amounts of orthophosphate, bicarbonate and tris(hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid.(2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KCl are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl)aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KCl, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrpancies occur with all of the other buffers mentioned.(3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KCl, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase.(4) We suggest that the protons produced by electron transport may be used directly for phosophorylation without ever entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.     

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers.

(1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.     

Cytoplasmic hydrogen ion diffusion coefficient.

The apparent cytoplasmic proton diffusion coefficient was measured using pH electrodes and samples of cytoplasm extracted from the giant neuron of a marine invertebrate. By suddenly changing the pH at one surface of the sample and recording the relaxation of pH within the sample, an apparent diffusion coefficient of 1.4 +/- 0.5 x 10(-6) cm2/s (N = 7) was measured in the acidic or neutral range of pH (6.0-7.2). This value is approximately 5x lower than the diffusion coefficient of the mobile pH buffers (approximately 8 x 10(-6) cm2/s) and approximately 68x lower than the diffusion coefficient of the hydronium ion (93 x 10(-6) cm2/s). A mobile pH buffer (approximately 15% of the buffering power) and an immobile buffer (approximately 85% of the buffering power) could quantitatively account for the results at acidic or neutral pH. At alkaline pH (8.2-8.6), the apparent proton diffusion coefficient increased to 4.1 +/- 0.8 x 10(-6) cm2/s (N = 7). This larger diffusion coefficient at alkaline pH could be explained quantitatively by the enhanced buffering power of the mobile amino acids. Under the conditions of these experiments, it is unlikely that hydroxide movement influences the apparent hydrogen ion diffusion coefficient.     

Modulation of flash-induced photosystem II fluorescence by events occurring at the water oxidizing complex.

The mechanism of flash-induced changes with a periodicity of four in photosystem II (PSII) fluorescence was investigated with the aim of further using fluorescence measurements as an approach to studying the structural and functional organization of the water-oxidizing complex (WOC). The decay of the flash-induced high fluorescence state of PSII was measured with pulse amplitude modulated fluorometry in thylakoids and PSII enriched membrane fragments. Calculated QA- decay was well described by three exponential decay components, reflecting QA- reoxidation with halftimes of 450 and 860 micros, 2 and 7.6 ms, and 111 and 135 ms in thylakoids and PSII membranes, respectively. The effect of modification of the PSII donor side by changing pH or by removal of the extrinsic 17 and 24 kDa proteins on period four oscillations in both maximum fluorescence yield and the relative contribution of QA- reoxidation reactions was compared to flash-induced oxygen yield. The four-step oxidation of the manganese cluster of the WOC was found to be necessary but not sufficient to produce modulation of PSII fluorescence. The capacity of the WOC to generate molecular oxygen was also required to observe a period four in the fluorescence; however, direct quenching by oxygen was not responsible for the modulation. Potential mechanisms responsible for the periodicity of four in both maximum fluorescence yield pattern and flash-dependent changes in proportion of centers with different QA- reoxidation rates are discussed with respect to intrinsic deprotonation events occurring at the WOC.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

Proton movements and electric potential generation in reconstituted ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716

ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes. The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 [Van Walraven et al. (1983) Eur. J. Biochem. 137, 101-106]. The native cartenoids and added oxonol VI served as probes for the electric membrane potential generated by the net charge separation (negative outside, positive inside). Their responses, with similar half-times as 9-tetradecylamino-6-chloro-2-methoxyacridine, are sensitive to valinomycin and stimulated by nigericin, as expected. The proton concentrations of extraliposomal and intraliposomal aqueous spaces were monitored by neutral red and cresol red; for internal measurements these pH indicators were trapped inside the vesicles during detergent dialysis. Internal acidification and external alkalinization induced by ATP hydrolysis are inhibited by nigericin and enhanced by valinomycin; at the commonly used higher valinomycin concentrations the neutral red response becomes transient, while the much slower cresol red response is diminished right from its onset. At smaller preset pH gradients both ATP hydrolysis activity and neutral red response are diminished. At increasing MgCl2 concentrations the neutral red responses are slowed down and the cresol red responses are slightly enhanced; this is observed for both internal and external dye responses. Neutral red permeation through the membrane is insignificant under our experimental conditions but is enhanced at temperatures below the lipid-phase transition. In the case of externally added neutral red the non-permeant buffer Hepes is only effective at high MgCl2 concentration, whereas some external cresol red response is visible only at high MgCl2 concentration in the presence of Hepes. The kinetics of the pH indicator and electric potential probe responses clearly distinguish fast interfacial and intra-membrane proton displacements from slow bulk proton equilibration. The data are summarized in a model that supports the importance of localized proton displacements for the primary energy-transducing events.     

The accumulation of neutral red in illuminated thylakoids

Thylakoids isolated from spinach (Spinacia oleracea L.) bind only a small fraction of neutral red in the dark whereas they accumulate large amounts of the protonated dye in their inner space under light. Light-induced neutral red uptake depends on the size of the proton gradient across the thylakoid membrane but does not follow the mechanism established for amines. Instead, the correlation between pH gradient and neutral red uptake can be predicted quantitatively assuming that protonated neutral red is accumulated mainly as dimer species.Under appropriate conditions, accumulation of protonated neutral red in the inner thylakoid space is proportional to an absorbance increase at 520 nm. This 520-nm change can be used for the continuous measurement of pH changes in thylakoids during steady-state illumination.     

Hemoglobins of the tadpole of the bullfrog, Rana catesbeiana. Temperature dependence of oxygen binding and pH dependence of subunit dissociation.

The temperature dependence of the oxygen equilibrium of tadpole hemoglobin has been determined between 0 degrees and 32 degrees for the unfractionated but phosphate-free lysate and between 12 degrees and 32 degrees for each of the four isolated components between pH 6 and 10 in 0.05 M cacodylate, Tris, or glycine buffers containing 0.1 M NaCl and 1 mM EDTA. Under these conditions the Bohr effect (defined as deltalog p50/deltapH) of the unfractionated lysate is positive at low temperatures between pH 6 and 8.5 and is negative above pH 8.5 to 8.8 at any temperature. As the temperature rises the Bohr effect below pH 8.5 changes greatly. In the interval pH 7.0 to 7.5, the magnitude of the Bohr effect decreases from + 0.28 at 0 degrees to zero at about 24 degrees and becomes negative, as in mammalian hemoglobins, above this temperature. Measurements with the isolated components show that the temperature dependence of oxygen binding for Components I and II and for Components III and IV is very similar. For both sets of components the apparent overall enthalpy of oxygenation at pH 7.5 is about -16.4 kcal/mol and -12.6 kcal/mol at pH 9.5. The measured enthalpies include contributions from the active Bohr groups, the buffer ions themselves, the hemoglobin groups contributing buffering, and any pH-dependent, oxygenation-dependent binding of ions such as chloride by the hemoglobin. The apportioning of the total enthalpy among these various processes remains to be determined. Between pH 8 and 10.5 tadpole oxyhemoglobin undergoes a pH-dependent dissociation from tetramer to dimer. The pH dependence of the apparent tetramer-dimer dissociation constant indicates that at pH 9.5 the dissociation of each tetramer is accompanied by the release of approximately 2 protons. In this pH range the oxygen equilibrium measurements indicate that about 0.5 proton is released for each oxygen molecule bound. The results are consistent with the conclusion that one acid group per alphabeta dimer changes its pK from about 10 to 8 or below upon dissociation of the tetramer.     

The secretory activity of algal cells in buffered media

Summary The effect of the activity of algal cells on the buffering characteristics of suspending fluid was studied onChlorella 7-11-05 cells suspended in Tris buffer or in sodium citrate-citric acid buffer. It was observed that changes in the buffering capacity of the suspending fluid depended on the concentration of the original buffer, pH, and the nature of the buffer.In Tris buffer, which is not consumed byChlorella cells, the buffering capacity, measured in terms of the van Slyke's buffer index (), increased with time. In sodium citrate-citric acid buffer, which is used byChlorella cells in the course of their metabolism, not only the buffer index () changed, but also the pH at which the suspending fluid had maximum buffering capacity shifted to a new value. In both Tris and citrate buffers the newly formed buffer systems had a maximum buffering capacity at pH 6.4 corresponding to the pK₁ of carbonic acid.This work was supported by funds from the National Aeronautics and Space Administration.Scientific Article A 1237, Contribution No. 3742 of the University of Maryland Agricultural Experiment Station.     

Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy.

Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196 degrees C were studied by various semiquantitative methods, including simple visual measurements of colour changes with pH indicators, as well as measurements of pH-dependent changes in the e.p.r. (electron paramagnetic resonance) spectra of solutions of three different metalloenzymes. It is concluded that apparent pH changes of up to about 3pH units may occur under particular conditions. Such changes were independent of the time taken to freeze the samples, when this was varied from about 3ms t0 20s, but were affected by the presence of some proteins in solution. Recommendations on the buffers that should be used to avoid such apparent pH changes in e.p.r. spectroscopy and other low-temperature biochemical work are made. Phosphate and pyrophosphate buffers, which gave large decreases (2-3 pH units), and Tris, which under some conditions gave increases of about the same magnitude, are to be avoided. Certain zwitterionic buffers such as Bicine [NN-bis-(2-hydroxyethyl)glycine] are satisfactory. Apparent pH effects were found to depend on buffer and protein concentration. It is therefore recommended that as a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed.     

The effect of pH on the growth of saprotrophic and ectomycorrhizal ammonia fungi in vitro

Some saprotrophic and ectomycorrhizal fungi produce reproductive structures, preferably in slightly alkaline to neutral forest soil. This research examines the growth of these "ammonia fungi" in liquid medium at various pH values. In the first experiment, the capacity of six buffers was examined to select appropriate buffers for stabilizing pH in the neutral-to-alkaline range by culture of three species of the ammonia fungi in media initially adjusted to pH 7, 8 or 9. The highest buffering capacity was shown in 2-(N-morpholino) ethanesulfonic acid (MES) at pH 7, and N, N-bis (2-hydroxyethyl) glycine (Bicine) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at pH 8 and 9. In the second experiment, the growth of 15 strains of both saprotrophic and ectomycorrhizal ammonia fungi was tested on the medium initially adjusted to pH 3, 4, 5, 6 or 7 with MES, or to pH 8 or 9 buffered with Bicine. Many of the saprotrophic species grew well at pH 7 or 8; the ectomycorrhizal species showed optimum growth at pH 5 or 6. The pH suitable for the in vitro growth of these fungi was correlated with the pH of forest soil where these fungi occur.     

Exposure of Leaves of Cucumis sativus L. to Low Temperatures in the Light Causes Uncoupling of Thylakoids II. Non-Destructive Measurements with Intact Leaves

To examine the effects of chilling of leaves of cucumber (Cucumissativus L.) in moderate light on the coupling state of thylakoidsin situ, changes in fluorescence, changes in light scatteringand flash-induced changes in absorbance at 518 nm were examinedin intact leaves. After chilling of leaves at 5?C in the lightfor 5 h, the non-photochemical quenching of fluorescence, ameasure of energisation of thylakoids, was largely suppressed.The treatment also caused a suppression of light-induced changesin the light scattering by leaves, which depends on the formationof a pH gradient across thylakoid membranes. When thylakoidswere prepared by very gentle methods from the leaves chilledin the light, through a step of preparation of intact chloro-plasts,the transport of electrons from H₂O to ferricyanide was uncoupled,being insensitive to an uncoupler, methylamine. These data provide consistent evidence that the thylakoids areuncoupled in situ by the chilling of leaves in the light and,as a consequence of the uncoupling, the energisation of themembranes is suppressed. However, the decay of the flash-inducedchange in absorbance at 518 nm in leaves was not markedly acceleratedby the treatment. The thylakoids isolated from leaves chilledin the light, which were in the uncoupled state, also did notshow a rapid decay, unless an efficient uncoupler such as gramicidinwas added. These results suggest that even a considerable uncouplingof thylakoids, brought about by chilling of leaves in the light,is not sufficient to cause a marked acceleration of the decayof the flash-induced change in absorbance at 518 nm. Therefore,analysis at 518 nm is not always a sensitive method for assessingthe coupling state of thylakoids. (Received July 1, 1991; Accepted October 4, 1991)     

The effects of elevated pH and high salt concentrations on tubulin

The effects of incubating phosphocellulose-purified bovine tubulin at 4 degrees C in nucleotide-free buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH4)2SO4, or NH4Cl have been studied. At pH greater than or equal to 7.5 or at NaCl concentrations greater than or equal to 0.7 M, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 M 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 M NH4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 M (NH4)2SO4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220-260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 mM guanosine 5'-triphosphate (GTP) or 4 M glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 M NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 M NaCl is decreased in the presence of 1 mM GTP, and tubulin is protected almost completely from 1 M NaCl-induced loss of GTP (and retains the ability to exchange [3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.