Internal pH indicators for biomolecular NMR

We describe a non-invasive technique for determining pH in biomolecular NMR sample using buffer components (formate, tris, piperazine, and imidazole) as internal pH indicators, whose (1)H NMR chemical shifts are sensitive to pH in a range from 2.5 to 9.8. This method is suitable for a wide range of applications where samples are handled intensively during NMR titrations or in high throughput analysis in structural genomics or metabolomics.     

Dicarboxy-dichlorofluorescein: a new fluorescent probe for measuring acidic intracellular pH

Derivatives of fluorescein sensitive to pH are extensively utilized for the determination of intracellular pH (pHi). Available dyes have pKa values of approximately 7.0, and are not well suited for measuring acidic pHi. We examined the fluorescein derivative, 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDCF) for its potential in the microspectrofluorometric measurement of pHi during acidic conditions. CDCF showed intense fluorescence and pH sensitivity near its "effective" pKa value of 4.2, using a 495/440 nm dual excitation wave-length ratio method. Protein interactions caused fluorescence ratio deviations which were most pronounced at the extremes of pH, whereas calcium and magnesium concentrations had little effect on the fluorescent ratio intensity. Intracellular calibration performed using nigericin in the presence of high potassium eliminated the need to correct for protein interactions, and the ratio method minimized any variations due to dye concentration differences or instrument fluctuation. Intracellular retention of the dye was high, and 95% of the initial signal remained after 1 h. Fluorescence bleaching was 14.5% after 1 h of continuous excitation and cell survival was not affected by dye loading. We conclude that CDCF is an excellent intracellular pH indicator in the pH range of 4-5.     

Geomicrobiology of extremely acidic subsurface environments

Extreme acidophiles (microorganisms with pH optima of 相似文献   

Quantization of pH: evidence for acidic activity of triglyceride lipases

Here we present a study of lipolytic activity of lipases from Fusarium solani pisi (cutinase), Rhizomucor miehei, Pseudomonas cepacia, and Humicola lanuginosa. Their activities toward triolein provide clear evidence for considerable enzymatic activity under acidic conditions. The activity was followed using Fourier transform infrared attenuated total reflection (FTIR-ATR) and nuclear magnetic resonance (NMR). Using these approaches, all the lipases that were studied exhibited lipolytic activity down to pH 4. The common model for the catalytic activity of the F. solani pisi cutinase, and lipases in general, requires the deprotonation of the active site histidine. Measurements using (13)C NMR spectroscopy showed a pK(a) value in the absence of substrate that is not consistent with the detected acid activity. We propose a novel model for the electrostatics in the active site of cutinase that could explain the observed acidic activity. The active site is essentially covered with the lipid surface during catalysis, thus preventing chemical communication between the active site and the bulk solvent. We propose that the classical definition of pH in bulk solution is not applicable to the active site environment of a lipase when the active site is inaccessible to solvent. In small restricted volumes, the pH must be quantized, and since much of the biological world is dependent on compartmentalization of processes in small volumes, it becomes relevant to investigate when this mechanism comes into play. We have made a quantitative assessment of how large the restricted volume can be and still lead to quantization of pH.     

The use of pH indicators to identify suitable environments for freezing samples in aqueous and mixed aqueous/nonaqueous solutions

The colours of frozen solutions containing pH indicators are shown to provide a test for changes in pH in the solvent environment which occur on freezing. Yeast alcohol dehydrogenase loses activity on freezing in phosphate buffer (a buffer in which pH indicator colour changes shows a marked decrease in pH on freezing) but when frozen in bis-tris, Hepes, or N-glycylglycine buffers (all of which show little change in the colour of universal pH indicator and hence of pH on freezing) is stable on freezing. The effects of freezing in different buffer systems on the rate of decomposition of NADPH, and on the rate hydrolysis of 4-nitrophenyl acetate, are rationalised in terms of the pH shifts in these buffers which were determined using universal pH indicator. It is proposed that a major reason for the instability of samples on freezing is the pH changes which occur when some systems are frozen. From the results a general scheme for selecting the best environment for safely freezing samples is proposed which is based on the use of pH indicators.     

Enhanced extracellular lipid accumulation in acidic environments

PURPOSE OF REVIEW: Binding of apolipoprotein B-100-containing lipoproteins (VLDL, IDL, and LDL) to proteoglycans and modifications of the lipoproteins, whether bound or unbound, are key processes in atherogenesis. The complex interplay between binding and modification has been studied at neutral pH conditions. It has been demonstrated that during atherogenesis the extracellular pH of the lesions decreases. We summarize findings suggesting that lipoprotein binding and modification are enhanced at acidic pH. RECENT FINDINGS: Many enzymes found in the arterial intima, such as secretory sphingomyelinase and cathepsins, are able to hydrolyze lipoproteins in vitro. These enzymes function optimally at slightly acidic pH (pH 5.5-6.5), and are likely to act on lipoproteins optimally in the acidic plaque areas. Also, the ability of human aortic proteoglycans to bind native VLDL, IDL, and LDL is dramatically increased at acidic pH; this binding can be further increased if these apolipoprotein B-100-containing particles are hydrolytically modified. SUMMARY: Recent in-vitro findings suggest that in areas of atherosclerotic arterial intima where the extracellular pH is decreased, binding of apolipoprotein B-100-containing lipoproteins to proteoglycans and modification of the lipoproteins by acidic enzymes are enhanced. The pH-induced amplification of these processes will lead to enhanced extracellular accumulation of lipoproteins and accelerated progression of the disease.     

Autotrophic,sulfur-oxidizing actinobacteria in acidic environments

Some novel actinobacteria from geothermal environments were shown to grow autotrophically with sulfur as an energy source. These bacteria have not been formally named and are referred to here as “Acidithiomicrobium” species, as the first of the acidophilic actinobacteria observed to grow on sulfur. They are related to Acidimicrobium ferrooxidans with which they share a capacity for ferrous iron oxidation. Ribulose bisphosphate carboxylase/oxygenase (RuBisCO) is active in CO₂ fixation by Acidimicrobium ferrooxidans, which appears to have acquired its RuBisCO-encoding genes from the proteobacterium Acidithiobacillus ferrooxidans or its ancestor. This lateral transfer of RuBisCO genes between a proteobacterium and an actinobacterium would add to those noted previously among proteobacteria, between proteobacteria and cyanobacteria and between proteobacteria and plastids. “Acidithiomicrobium” has RuBisCO-encoding genes which are most closely related to those of Acidimicrobium ferrooxidans and Acidithiobacillus ferrooxidans, and has additional RuBisCO genes of a different lineage. 16S rRNA gene sequences from “Acidithiomicrobium” species dominated clone banks of the genes extracted from mixed cultures of moderate thermophiles growing on copper sulfide and polymetallic sulfide ores in ore leaching columns.     

Gramicidin derivatives as membrane-based pH sensors

Ion channels provide a means for sensitive pH measurement at membrane interfaces. Detailed knowledge of the structure and function of gramicidin channels permits the engineering of pH-sensitive derivatives. Two derivatives, gramicidin-ethylenediamine and gramicidin-histamine, are shown to exhibit pH-dependent single-channel behaviour over the pH ranges 9-11 and 6.5-8.5, respectively. Thermal isomerization of a carbamate group at the entrance of the channels leads to a pattern of steps in single-channel recordings. The size of the steps depends on the time-averaged degree of protonation of the appended group (ethylenediamine or histamine). Measurement of the size of the steps thus permits single-molecule pH sensing under symmetrical pH conditions or in the presence of a pH gradient.     

Genetically encoded fluorescent indicators for live cell pH imaging

Background

Intracellular pH underlies most cellular processes. There is emerging evidence of a pH-signaling role in plant cells and microorganisms. Dysregulation of pH is associated with human diseases, such as cancer and Alzheimer's disease.

Eukaryotes in acidic mine drainage environments: potential applications in bioremediation

Passive remediation of Acid Mine Drainage (AMD) is a popular technology continuously under development for more than 50 years now. Roles of eukaryotes, the natural residents of AMD and its attenuator are not emphasized adequately. Studies suggest that macrophyte distinctively generate alkalinity through benthic sediments as part of root respiration. Other eukaryotic populations effectively enrich the carbon source for maintaining sulphate reducing bacterial (SRB) populations and act symbiotically. Algae produce anoxic zones for SRB action and help in biogenic alkalinity generation. While studies on algal populations and actions are relatively available those on fungal population are limited. Fungi show capacity to absorb significant amount of metals in their cell wall, or by extra cellular polysaccharide slime. This review examines the roles of these microorganisms and documents their activities in holistic form in the mine water environment. This work discusses the potential areas of likely future research that could enable AMD remediation using active eukaryotes to be made on sound understanding.     

New class of 19F pH indicators: fluoroanilines

The pH dependence of the 19F chemical shift has been characterized for a number of fluorine-substituted aniline derivatives. These compounds constitute a new class of 19F nuclear magnetic resonance (NMR) pH indicators, characterized by single 19F resonance lines with sensitivities ranging from 2 to 7 ppm/pH unit near the aniline pKa; total shifts between conjugate acid and base of 5-15 ppm; and pKas ranging from 1 to 7. One compound, N,N-(methyl-2-carboxyisopropyl)-4-fluoroaniline, has a pKa of 6.8 and a sensitivity of 5 ppm/pH unit. This compound displays significant broadening of its 19F resonance near the aniline pKa (6.8), due to a decreased rate of exchange between conjugate acid and base species. Our results are consistent with slow dissociation of an intramolecular hydrogen bond in the zwitterionic species that limits the exchange rate between protonated and unprotonated forms for N,N-(methyl-2-carboxyisopropyl)-4-fluoroaniline.     

Modulation of TRPM2 by acidic pH and the underlying mechanisms for pH sensitivity

TRPM2 is a Ca²⁺-permeable nonselective cation channel that plays important roles in oxidative stress–mediated cell death and inflammation processes. However, how TRPM2 is regulated under physiological and pathological conditions is not fully understood. Here, we report that both intracellular and extracellular protons block TRPM2 by inhibiting channel gating. We demonstrate that external protons block TRPM2 with an IC₅₀ of pH_o = 5.3, whereas internal protons inhibit TRPM2 with an IC₅₀ of pH_i = 6.7. Extracellular protons inhibit TRPM2 by decreasing single-channel conductance. We identify three titratable residues, H958, D964, and E994, at the outer vestibule of the channel pore that are responsible for pH_o sensitivity. Mutations of these residues reduce single-channel conductance, decrease external Ca²⁺ ([Ca²⁺]_o) affinity, and inhibit [Ca²⁺]_o-mediated TRPM2 gating. These results support the following model: titration of H958, D964, and E994 by external protons inhibits TRPM2 gating by causing conformation change of the channel, and/or by decreasing local Ca²⁺ concentration at the outer vestibule, therefore reducing [Ca²⁺]_o permeation and inhibiting [Ca²⁺]_o-mediated TRPM2 gating. We find that intracellular protons inhibit TRPM2 by inducing channel closure without changing channel conductance. We identify that D933 located at the C terminus of the S4-S5 linker is responsible for intracellular pH sensitivity. Replacement of Asp⁹³³ by Asn⁹³³ changes the IC₅₀ from pH_i = 6.7 to pH_i = 5.5. Moreover, substitution of Asp⁹³³ with various residues produces marked changes in proton sensitivity, intracellular ADP ribose/Ca²⁺ sensitivity, and gating profiles of TRPM2. These results indicate that D933 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM2 channel gating. Collectively, our findings provide a novel mechanism for TRPM2 modulation as well as molecular determinants for pH regulation of TRPM2. Inhibition of TRPM2 by acidic pH may represent an endogenous mechanism governing TRPM2 gating and its physiological/pathological functions.     

Ecophysiology of algae living in highly acidic environments

Highly acidic environments are inhabited by acidophilic as well as acidotolerant algae. Acidophilic algae are adapted to pH values as low as 0.05 and unable to grow at neutral pH. A prerequisite for thriving at low pH is the reduction of proton influx and an increase in proton pump efficiency. In addition, algae have to cope with a limited supply of carbon dioxide for photosynthesis because of the absence of a bicarbonate pool. Therefore, some algae grow mainly in near terrestrial situations to increase the CO₂-availability or actively move within the water body into areas with high CO₂. Beside these direct effects of acidity, high concentrations of heavy metals and precipitation of nutrients cause indirect effects on the algae in many acidic environments.     

Gramicidin derivatives as membrane-based pH sensors.

Ion channels provide a means for sensitive pH measurement at membrane interfaces. Detailed knowledge of the structure and function of gramicidin channels permits the engineering of pH-sensitive derivatives. Two derivatives, gramicidin-ethylenediamine and gramicidin-histamine, are shown to exhibit pH-dependent single-channel behaviour over the pH ranges 9-11 and 6.5-8.5, respectively. Thermal isomerization of a carbamate group at the entrance of the channels leads to a pattern of steps in single-channel recordings. The size of the steps depends on the time-averaged degree of protonation of the appended group (ethylenediamine or histamine). Measurement of the size of the steps thus permits single-molecule pH sensing under symmetrical pH conditions or in the presence of a pH gradient.     

Self-assembly of laminin induced by acidic pH

The supramolecular architecture of the basement membrane is provided by two enmeshed networks of collagen IV and laminin. The laminin network is maintained exclusively by interactions among individual laminin molecules and does not depend on the presence of other extracellular matrix components. Laminin polymers can be obtained in vitro either in solution or in association with the surface of bilayers containing acidic lipids. In this work, we have tested the hypothesis that the negative charges present on acidic lipids establish an acid microenvironment that is directly responsible for inducing laminin aggregation. Using light-scattering measurements, we show that laminin does not aggregate on vesicles of neutral lipids, whereas instantaneous aggregation occurs to progressively greater extents as the proportion of acidic phospholipids in the vesicles is increased. Aggregation of laminin induced by vesicles containing acidic phospholipids occurs very rapidly, so that maximal aggregation for each condition is reached within 1 min after laminin dilution. Aggregation depends on the presence of Ca(2+) ions, is reversed by increasing ionic strength, and can be detected at laminin concentrations as low as 6 nM. In addition, we show that, in the absence of vesicles, acidification of the bulk solution can also induce laminin self-polymerization through a process that exhibits the same properties as lipid-induced polymerization. The fact that there is a correspondence between the processes of self-polymerization of laminin in acidic medium and in neutral medium but in the presence of vesicles containing negatively charged lipids leads us to propose that the microenvironment of an acidic surface may trigger the assembly of laminin networks. In vivo, such an acidic microenvironment would be provided by negatively charged sialic acid and sulfate groups present in the glycocalyx surrounding the cells.     

Reversible inactivation of serpins at acidic pH

The inhibitory activity of the serpins alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, alpha(2)-antiplasmin, antithrombin and C(1)-esterase inactivator is rapidly lost at pH 3 but slowly recovers at pH 7.4 with variable first-order rates (t(1/2)=1.4-19.2 min). All except alpha(1)-antichymotrypsin undergo a variation in intrinsic fluorescence intensity upon acidification (midpoint ca. 4.5) with a slow bi-exponential return to the initial intensity at pH 7.4 (mean t(1/2)=2.3-23 min). No correlation was found between the time of fluorescence recovery and that of reactivation. The acid-treated serpins are proteolyzed at neutral pH by their target proteinases. alpha(1)-Proteinase inhibitor was studied in more detail. Its acidification at pH 3 has a mild effect on its secondary structure, strongly disorders its tertiary structure, changes the microenvironment of Cys(232) and causes a very fast change in ellipticity at 225 nm (t(1/2)=1.6s). Neutralization of the acid-treated alpha(1)-proteinase inhibitor is an exothermic phenomenon. It leads to a much faster recovery of activity (t(1/2)=4+/-1 min) than of fluorescence intensity (t(1/2)=23+/-19 min), ellipticity (t(1/2)=32+/-4 min) and change in total energy, indicating that the inhibitory activity of alpha(1)-proteinase inhibitor does not require a fully native structure.