The formulation of buffers and media for enzyme histochemistry

Summary Buffer solutions and incubating media for enzyme histochemistry are discussed in terms of pH, ionic strength and buffering capacity. A specially written program is presented. This program enables (i) buffers and media of known pH and ionic strength to be formulated; (ii) the ionic strength of a buffer solution of known molarity, and (iii) the thermodynamic acid dissociation constant of a buffer substance, to be calculated.     

The acylation of L-glycerol-3-phosphate by microsomes from rat brain: effects arising from the properties of the reaction medium

Abstract— The velocity of the reaction catalysed by acyl-CoA: l -giycerol-3-phosphate acyltransferase (EC 2.3.1.15) of microsomes from rat brain was affected by the nature of the buffering agent, the ionic strength and the sucrose concentration of the reaction medium. The enzyme was inhibited by buffers based on trimethyl-pyridine, diethyl barbituric acid, and boric acid. Buffers based on N-ethyl morpholine, potassium phosphate, sodium arsenate, imidazole, tris and triethanolamine were not inhibitory. Dithiothreitol protected the enzyme and produced maximal activity at levels in the reaction medium between 0.2 and 2.8 mM. Optimum ionic strength was determined by varying the concentration of a potassium phosphate buffer and in this medium the optimum ionic strength was about 0.2 M. In other studies with sodium formate, potassium acetate and other salts there was a broad plateau of activity in a range about 0.2 M. A study of pH vs. activity with two different buffering agents at constant ionic strength showed a broad maximum of activity from pH 7.2 to pH 7.8. The velocity of the reaction could be further increased by the inclusion of 0.25 M-sucrose in the reaction medium in the presence of 0.2 M salts. The sucrose effect produced maximum velocities at sucrose concentrations ranging from 0.2 to 0.6 M. The studies reported here indicate that the activity of the enzyme is dependent upon the state of hydration of the microsomal membranes and in part on the ability of the enzyme or membrane to cope with large micelles of S-palmityl-CoA.     

Do sperm contribute to the buffering capacity of steelhead trout (Oncorhynchus mykiss) semen?

The motility of salmonid sperm is pH-sensitive and the buffering capacity of the seminal plasma is low. The objective of the present study was to determine the extent to which sperm contribute to the buffering capacity of whole steelhead (Oncorhynchus mykiss) semen. To determine the buffering capacity, semen and seminal plasma samples were titrated with HCl and pH measurements taken at 1-2 min. The buffering capacity of semen was not different from that of seminal plasma over the pH range 7.5 to 8.5 and was approximately 15% to 20% less over the range 6.0 to 7.0. Comparable results were obtained for the semen and seminal plasma of the chinook salmon (Oncorhynchus tshawytscha). To assess whether the intracellular environment could influence the buffering capacity, the effects of cell disruption with n-butanol and Triton X-100 (TX-100) were determined. Over the pH range 7.5 to 8.5, the presence of n-butanol or TX-100 resulted in a doubling of the buffering capacity of the semen; TX-100, but not n-butanol, increased semen buffering capacity over the pH range 6.0 to 7.0. To determine whether the sperm's intracellular compartment might contribute to the buffering capacity over a longer duration, semen and seminal plasma samples were acidified with HCl and the pH measured over several hours. These data suggest that intact sperm contribute no more than about 25% to the buffering capacity of whole semen. The buffering capacity of steelhead semen and seminal plasma were comparably and modestly temperature sensitive. The results suggest that the sperm may contribute to the buffering capacity of the semen over a physiological pH range, however, if so, the effect is relatively small.     

Paleolimnological reconstruction of the effects of atmospheric deposition of acids and heavy metals on the chemistry and biology of lakes in New England and Norway

Sediment cores from nine lakes in southern Norway (N) and six in northern New England (NE) were dated by ¹³⁷Cs, ²¹⁰Pb and in NE also by pollen, and were analyzed geochemically and for diatoms. Cores from two N and three NE lakes were analyzed for cladocerans. ¹³⁷Cs dating is unreliable in these lakes, probably due to mobility of Cs in the sediment. In Holmvatn sediment, an up-core increase in Fe, starting ca. 1900, correlates with geochemical indications of decreasing mechanical erosion of soils. Diatoms indicate a lake acidification starting in the 1920's. We propose that soil Fe was mobilized and runoff acidified by acidic precipitation and/or by soil acidification resulting from vegetational succession following reduced grazing. Even minor land use changes or disturbances in lake watersheds introduce ambiguity to the sedimentary evidence relating to atmospheric influences. Diatom counts from surface sediments in 36 N and 31 NE lakes were regressed against contemporary water pH to obtain coefficients for computing past pH from subsurface counts. Computed decreases of 0.3–0.8 pH units start between I890 and I930 in N lakes already acidic (pH 5.0–5.5) before the decrease. These and lesser decreases in other lakes start decades to over a century after the first sedimentary indications of atmospheric heavy metal pollution. It is proposed that the acidification of precipitation accompanied the metal pollution. The delays in lake acidification may be due to buffering by the lakes and watersheds. The magnitude of acidification and heavy metal loading of the lakes parallels air pollution gradients. Shift in cladoceran remains are contemporary with acidification, preceding elimination of fishes.     

Tracking Recovery Patterns in Acidified Lakes: A Paleolimnological Perspective

Long-term data are often lacking to effectively assess patterns of lake acidification and recovery. Fortunately, paleolimnological techniques can be used to infer past changes in lakewater acidity and related variables by means of biological indicators, such as diatom valves and chrysophyte scales, preserved in ²¹⁰Pb-dated sediment cores. We summarize paleolimnological data that we have gathered from 36 Sudbury (Ontario) and 20 Adirondack Park (New York) lakes to estimate the magnitude of lake acidification and any subsequent recovery in these lake systems. In both regions, many lakes were shown to have acidified considerably, some over two pH units, since the 1850s. Although some recovery was noted in both lake regions, Sudbury lakes generally showed larger increases in inferred lakewater pH with recent declines in sulfur emissions. Possible explanations of these differences include the greater decrease in sulfate deposition in the Sudbury area, as well as generally longer residence times of lakes in Sudbury, perhaps allowing for more in-lake alkalinity generation. In addition, Sudbury lakes generally had higher pre-industrial pH levels, suggesting that lakes with higher natural buffering capacities are more likely to recover more quickly with declines in deposition, even if they had been acidified to a great extent.     

An alkaline approach to treating cooling towers for control of Legionella pneumophila

Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat.     

An alkaline approach to treating cooling towers for control of Legionella pneumophila.

Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat.     

pH changes in the rhizosphere in relation to the pH-buffering of soils

Summary pH values in the rhizosphere of peanut seedlings were measured with Sb micro electrodes in soils with a bulk pH between 3.9 and 7.7. Within 2–2.5 days the roots decreased soil pH to a minimum value which was linearly correlated with short term buffering capacity of the soils as determined by addition of HCl to soil suspensions. The greatest distance from the root surface where pH dropped significantly was found in soils with pH near 6. This maximum distance was also correlated with pH-buffering. The low buffering capacity for soils of pH near 6 seems to be valid for a large number of soils with different properties.     

pH gradients generated by polyprotic buffers. II. Experimental validation

The experimental validation refers to the computer program reported in the companion paper, able to simulate the course of pH, buffering power (beta) and ionic strength (I) of polyprotic buffers (either singly or in a mixture) titrated over any pH range. With simple oligoamines (up to five nitrogens) it is shown that it is impossible to generate linear pH gradients in the pH 4-10 interval, unless they are mixed in appropriate ratios. With pentaethylene hexamine, when used alone, it is possible to create a linear pH 4-10 interval, provided the molarity ratios are altered in the two chambers of the gradient mixer. The general rule operating for generation of linear pH intervals is constancy of buffering power throughout the titration. Local minima of beta produce steeper gradients, while local beta maxima flatten it. The ideal delta pK to arrange for linear pH gradients during titration is centred around 1 pH unit; thus polyprotic buffers with very large delta pK values (e.g., EDTA) appear to be totally useless for this purpose. The present computing algorithms should be quite efficient for optimizing existing buffer recipes for chromatofocusing or ampholyte displacement chromatography or for creating new, properly tailored, buffer mixtures.     

Buffer capacity of biologics—from buffer salts to buffering by antibodies

Controlling pH is essential for a variety of biopharmaceutical process steps. The chemical stability of biologics such as monoclonal antibodies is pH‐dependent and slightly acidic conditions are favorable for stability in a number of cases. Since control of pH is widely provided by added buffer salts, the current study summarizes the buffer characteristics of acetate, citrate, histidine, succinate, and phosphate buffers. Experimentally derived values largely coincide with values calculated from a model that had been proposed in 1922 by van Slyke. As high concentrated protein formulations become more and more prevalent for biologics, the self‐buffering potential of proteins becomes of relevance. The current study provides information on buffer characteristics for pH ranges down to 4.0 and up to 8.0 and shows that a monoclonal antibody at 50 mg/mL exhibits similar buffer capacity as 6 mM citrate or 14 mM histidine (pH 5.0–6.0). Buffer capacity of antibody solutions scales linearly with protein concentration up to more than 200 mg/mL. At a protein concentration of 220 mg/mL, the buffer capacity resembles the buffer capacity of 30 mM citrate or 50 mM histidine (pH 5.0–6.0). The buffer capacity of monoclonal antibodies is practically identical at the process relevant temperatures 5, 25, and 40°C. Changes in ionic strength of ΔI=0.15, in contrast, can alter the buffer capacity up to 35%. In conclusion, due to efficient self‐buffering by antibodies in the pH range of favored chemical stability, conventional buffer excipients could be dispensable for pH stabilization of high concentrated protein solutions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 480–492, 2013     

RELATIONSHIPS OF SEDIMENTED DIATOM SPECIES (BACILLARIOPHYCEAE) TO ENVIRONMENTAL GRADIENTS IN DILUTE NORTHERN NEW ENGLAND LAKES1

Limnological gradients of small, oligotrophic, and low conductance lakes in northern New England were defined by principal components analysis; relationships of sedimented diatom species to the gradients were investigated by correlation analysis. Diatom distributions were most strongly related to the gradient of pH and alkalinity and the covarying variables, conductance, Mg, Ca, total Al, and exchangeable Al. Weaker relationships to lake morphology, dissolved organic carbon and water color, altitude and marine aerosol inputs, and the distinctive water chemistry of some New Hampshire lakes were also present. Results for 16 taxa of importance in our studies of lake acidity are given in detail and are compared to results from other regions of eastern North America. Planktonic taxa were absent below pH 5.5, with the exception of the long form of Asterionella ralfsii var. americana Korn. The two forms of this taxon differed ecologically: the long form (>45μm) had an abundance weighted mean (AWM) pH 4.90 and occurred mostly in lakes that were deep relative to transparency; the short form (<45μm)had an AWM pH and occurred on lakes that were shallow relative to transparency. The ecological advantage of a “splitter” approach to diatom taxonomy was demonstrated by examination of other taxa as well, including Tabellaria flocculosa (Roth) Kütz. These results have important implications for paleolimnological interpretations.     

Environmental factors influencing zooplankton species composition of lakes in north-central Ontario,Canada

To assess the relative importance of lake chemistry, morphometry and zoogeography on limnetic zooplankton, we collected zooplankton, water, and morphometric data from 132 headwater Canadian Shield lakes in 6 regions across north-central Ontario. A subset of these lakes (n = 52) were fished with gill nets. We clustered lakes based on their zooplankton species composition (presence/absence). Discriminant analysis was employed to determine how well lake characteristics could predict zooplankton community types. Correct classification of zooplankton communities for three models ranged from 72 to 91%. Lake size, lake location, and buffering capacity were ranked as the most important factors separating lake groups. Fish abundance (CPUE) was not significant in distinguishing between zooplankton communities. Though the range of lake sizes was limited (1–110 ha), larger lakes tended to support more species. Lake location (zoogeography) also influenced species composition patterns. Although Algoma lakes tended to be larger (\-x = 18.0 ha, other lakes \-x = 2.5 ha), they supported relatively depauperate zooplankton communities. Buffering capacity was ranked third in the discriminant analysis models, but pH and alkalinity were not significantly different between lake groups.     

The temperature and pH dependence of conformational transitions of the chromatin subunit.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.     

A re-valuation of the Araphidineae/Centrales index as an indicator of lake trophic status

SUMMARY. Comparisons of pre- and post-settlement diatom assemblages from the sediment of twenty-five Minnesota lakes reveals that Stephanodiscus hantzschii percentages are more consistent indicators of human disturbance than the Araphidineae/Centrales (A/C) index. In a set of eighty surface sediment samples from lakes whose water chemistry is known, S. hantzschii , a centric diatom, is abundant as a microfossil in lakes with total phosphorus > 15 μg I⁻¹ and alkalinity > 1.5 m-equiv. I⁻¹. High Araphidineae/Centrales indices are characteristic of lakes with only moderate total phosphorus levels and very low alkalinities.     

The effects of pH on hyaluronate as observed by light scattering

Hyaluronate was investigated over a wide pH range, and at near zero and intermediate ionic strength, using dynamic and total intensity light scattering. Commercially obtained rooster comb hyaluronate was purified, and solutions were prepared in pure water by low-power bath ultrasonication and subsequent filtering. These solutions were of low polydispersity and appeared to contain single molecules of hyaluronate. Despite the absence of added electrolyte, these solutions yielded well-behaved Zimm plots. Increasing ionic strength and changing pH decreased radii of gyration and increased diffusion constants. Except for what appeared to be slow hydrolysis at either extreme of pH, molecular weights remained constant under all pH and ionic strength conditions. Under all solvent conditions investigated, diffusion coefficients increased with decreasing hyaluronate concentration. Unsonicated, lightly centrifuged solutions without added electrolyte were polydisperse, and their light scattering intensity was dominated by what appeared to be stable hyaluronate aggregates. The results are interpreted in terms of the polyelectrolyte properties of hyaluronate and its tendency to form stable entanglements, especially at low ionic strength. Previous light scattering studies in the literature on hyaluronate have shown widely varying results. The present article briefly reviews this literature and attempts to explain the variation among the previous results, emphasizing the Kuhn statistical segment length as an indicator of whether results are influenced by polydispersity or contaminants causing hyaluronate aggregation.     

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.     

Buffering capacity of bacilli that grow at different pH ranges.

Cytoplasmic buffering capacities and buffering by whole cells were examined in six bacterial species: Bacillus acidocaldarius, Bacillus stearothermophilus, Escherichia coli, Bacillus subtilis, Bacillus alcalophilus, and Bacillus firmus RAB. Acid-base titrations were conducted on whole cells and cells permeabilized with Triton X-100 or n-butanol. In all of the species examined, the buffering capacity of intact cells was generally a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied from species to species. Over the entire range of pH values from 4 to 9.5, B. subtilis exhibited a cytoplasmic buffering capacity that was much higher than that of B. stearothermophilus, B. acidocaldarius, or E. coli. The latter three species had comparable cytoplasmic buffering capacities at pH 4 to 9.5, as long as optimal conditions for cell permeabilization were employed. All of the nonalkalophiles exhibited a decrease in cytoplasmic buffering capacity as the external pH increased from pH 5 to 7. At alkaline pH values, the two thermophiles in the study had particularly low cytoplasmic buffering capacities, and the two alkalophilic bacteria had appreciably higher cytoplasmic buffering capacities than any of the other species studied. Cytoplasmic buffering capacities as high as 1,100 nmol of H+ per pH unit per mg of protein were observed in alkalophilic B. firmus RAB. Since previous studies have shown that immediate cytoplasmic alkalinization occurs upon loss of the active mechanisms for pH homeostasis in the alkalophiles, the very high buffering capacities apparently offer no global protection of internal pH. Perhaps, the high buffering capacities reflect protective mechanisms for specific macromolecules or process rather than part of the mechanisms for bulk pH homeostasis.     

Gelation of globular proteins: effect of pH and ionic strength on the critical concentration for gel formation. A simple model and its application to beta-lactoglobulin heat-induced gelation.

The influence of pH (2-9) and ionic strength (0-0.14 M NaCl) on the sol-gel transition of beta-lactoglobulin was investigated in order to determine the critical gel concentration (C0). The concentration necessary to form a gel near the isoelectric pH remains approximately constant (approximately 1% w/v) independently of the ionic strength. At other pH values, the higher the ionic strength is, the lower the protein concentration must be to form a gel. A theoretical model to relate the effect of the intensity and the range of electrostatic interactions on the critical concentration (C0) is proposed and fits reasonably with the experimental results.     

Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency

Control of acidity and preventing intracellular acidosis are recognized as critical properties of an effective organ preservation solution. Buffer capacity and efficiency are therefore important for comparing the relative merits of preservation fluids for optimum hypothermic storage, but these parameters are not available for the variety of organ preservation solutions of interest in transplantation today. Moreover, buffer capacity is dependent upon both concentration and pH such that buffer capacity is not easily predicted for a complex solution containing multiple buffer species. Using standard electrometric methods to measure acid dissociation constants, this study was undertaken to determine the maximum and relative buffer capacities of a variety of new and commonly used hypothermic preservation solutions as a function of temperature. The reference data provided by these measurements show that comparative buffer capacity and efficiency vary widely between the commonly used solutions. Moreover, the fluids containing zwitterionic sulfonic acid buffers such as Hepes possess superior buffering for alpha-stat pH regulation in the region of physiological importance.     

Buffering capacity and membrane H+ conductance ofZymomonas mobilis

Buffering power and membrane conductance to H⁺ were measured inZymomonas mobilis subspmobilis ATCC 29191 by a pulse technique. Over the pH range studied, from 4.02 to 7.44,Z. mobilis presented very high values of cytoplasmic buffering capacity; it was a significant proportion of the total buffering capacity. These results support the idea that the cytoplasmic buffering power might be part of the pH homeostatic mechanism.