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.     

Role of amylase, mucin, IgA and albumin on salivary protein buffering capacity: A pilot study

It has been suggested that proteins serve as major salivary buffers below pH?5. It remains unclear, however, which salivary proteins are responsible for these buffering properties. The aim of this pilot study was to evaluate the correlation between salivary concentration of total protein, amylase, mucin, immunoglobulin A (IgA), albumin and total salivary protein buffering capacity at a pH range of 4–5. In addition, the buffering capacity and the number of carboxylic acid moieties of single proteins were assessed. Stimulated saliva samples were collected at 9:00, 13:00 and 17:00 from 4 healthy volunteers on 3 successive days. The buffering capacities were measured for total salivary protein or for specific proteins. Also, the concentration of total protein, amylase, mucin, IgA and albumin were analysed. Within the limits of the current study, it was found that salivary protein buffering capacity was highly positively correlated with total protein, amylase and IgA concentrations. A weak correlation was observed for both albumin and mucin individually. Furthermore, the results suggest that amylase contributed to 35% of the salivary protein buffering capacity in the pH range of 4–5.     

Calretinin: Modulator of neuronal excitability

Calretinin is a member of the calcium-binding protein EF-hand family first identified in the retina. As with the other 200-plus calcium-binding proteins, calretinin serves a range of cellular functions including intracellular calcium buffering, messenger targeting, and is involved in processes such as cell cycle arrest, and apoptosis. Calcium-binding proteins including calretinin are expressed differentially in neuronal subpopulations throughout the vertebrate and invertebrate nervous system and their expression has been used to selectively target specific cell types and isolate neuronal networks. More recent experiments have revealed that calretinin plays a crucial role in the modulation of intrinsic neuronal excitability and the induction of long-term potentiation (LTP). Furthermore, selective knockout of calretinin in mice produces disturbances of motor coordination and suggests a putative role for calretinin in the maintenance of calcium dynamics underlying motor adaptation.     

Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (Delta(pH)), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

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.     

Amphoteric, buffering chromatographic beads for proteome prefractionation. I: theoretical model

The possibility is reported here of fractionating proteins on amphoteric, buffering resins via ion-exchange chromatography. A given protein's adsorption to a particular amphoteric buffering resin is characterized by a bell-shaped curve in which the maximum protein binding capacity is observed at an optimum pH value lying approximately midway between the isoelectric point values (pI) of the resin and the protein. On either side of this maximum the protein binding capacity declines steadily, reaching zero at the pI of either the protein or exchanger. For instance, on beads of pI equal to 8, four proteins, two acidic (bovine albumin and ovalbumin) and two basic (cytochrome c and lysozyme), exhibit binding curves reaching zero values for the whole set when the exchanger is conditioned at pH 8.0. Away from the pI, and on both sides of the pH scale, the bell-shaped adsorption curves reach a maximum, for each protein, at a pH located at the midpoint between the pI values of each protein and that of the exchanger, and decline steadily to reach zero at the pI value of each protein species. Separation of model proteins using different amphoteric buffering resins of various pI was possible at different pH values according to both the pI of the proteins and of the exchangers. It was also demonstrated, using surface enhanced laser desorption/ionization mass spectrometry and two dimensional electrophoretic mapping, that separation of an Escherichia coli cell lysate on columns packed with amphoteric buffering resins of different pI and titrated to a particular pH value, delivered two distinctly different fractions, i.e. characteristically composed of, on the one hand, proteins having a pI below the buffer pH (the 'adsorbed' fraction), and on the other, of alkaline proteins possessing a pI above the pH of the buffer (the 'unadsorbed' fraction). This approach represents an attractive addition and/or alternative to the armory of protein pre-fractionation techniques currently employed in proteomics.     

Evolutionary Capacitance and Control of Protein Stability in Protein-Protein Interaction Networks

In addition to their biological function, protein complexes reduce the exposure of the constituent proteins to the risk of undesired oligomerization by reducing the concentration of the free monomeric state. We interpret this reduced risk as a stabilization of the functional state of the protein. We estimate that protein-protein interactions can account for of additional stabilization; a substantial contribution to intrinsic stability. We hypothesize that proteins in the interaction network act as evolutionary capacitors which allows their binding partners to explore regions of the sequence space which correspond to less stable proteins. In the interaction network of baker''s yeast, we find that statistically proteins that receive higher energetic benefits from the interaction network are more likely to misfold. A simplified fitness landscape wherein the fitness of an organism is inversely proportional to the total concentration of unfolded proteins provides an evolutionary justification for the proposed trends. We conclude by outlining clear biophysical experiments to test our predictions.     

Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes.

Intracellular passive Ca2+, buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 microM (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler "1799" was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+]i) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+]i during the pulse. In the range of [Ca2+]i from 0.1 to 2 microM, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, KD, of of 0.63 +/- 0.07 microM (n = 5) and a binding capacity, Bmax, of 162 +/- 15 mumol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of KD = 0.96 +/- 0.18 microM and Bmax = 123 +/- 18 mumol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+]i from 0.1 to 1 microM during a contraction requires approximately 50 microM Ca2+ to be added to the cytosol.     

Modelling intracellular H(+) ion diffusion

Intracellular pH, an important modulator of cell function, is regulated by plasmalemmal proteins that transport H(+), or its equivalent, into or out of the cell. The pH(i) is also stabilised by high-capacity, intrinsic buffering on cytoplasmic proteins, oligopeptides and other solutes, and by the extrinsic CO(2)/HCO(3)(-) (carbonic) buffer. As mobility of these buffers is lower than for the H(+) ion, they restrict proton diffusion. In this paper we use computational approaches, based on the finite difference and finite element methods (FDM and FEM, respectively), for analysing the spatio-temporal behaviour of [H(+)] when it is locally perturbed. We analyse experimental data obtained for various cell-types (cardiac myocytes, duodenal enterocytes, molluscan neurons) where pH(i) has been imaged confocally using intracellular pH-sensitive dyes. We design mathematical algorithms to generate solutions for two-dimensional diffusion that fit data in terms of an apparent intracellular H(+) diffusion coefficient, D(H)(app). The models are used to explore how the spatial distribution of [H(+)](i) is affected by membrane H(+)-equivalent transport and by cell geometry. We then develop a mechanistic model, describing spatio-temporal changes of [H(+)](i) in a cardiac ventricular myocyte in terms of H(+)-shuttling on mobile buffers and H(+)-anchoring on fixed buffers. We also discuss how modelling may include the effects of extrinsic carbonic-buffering. Overall, our computational approach provides a framework for future analyses of the physiological consequences of pH(i) non-uniformity.     

Cerebral Acid Buffering Capacity at Different Ages Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6-6.0, compared to 7.0-6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1-month-old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.     

Cytosolic pH Variations in Perfused Rat Liver at 4°C: Role of Intracellular Buffering Power

The effect of low temperature on cytosolic pH regulation and buffering capacity was evaluated in the isolated rat liver. The pH changes were followed by phosphorus-31 nuclear magnetic resonance. Cooling from 37 to 4°C, with Krebs–Heinseleit perfusion at an external pH of 7.35, induced an alkaline shift in cytosolic pH (pH_cyt) of 0.13 or 0.75 pH units in the presence or absence of bicarbonate, respectively (dpH_cyt/dT values were 0.004 and 0.022 unit/°C). With 4°C perfusion, in the presence or absence of bicarbonate, acute changes of external pH (from 7.40 to 5.90) did not affect pH_cyt. In contrast, intracellular loading with isobutyric acid or NH₄Cl induced rapid pH_cytvariations. The intrinsic buffering power value (10 to 50 slykes) measured in the absence of bicarbonate depended on pH_cyt. The larger value was observed for pH_cyt7.30, a value near the pK value of the imidazole group of intracellular proteins at 4°C. The presence of bicarbonate modified the amplitude of the pH_cytchange by increasing the total buffering power. It was demonstrated that during hypothermia, ionic carriers are inactivated and the charged forms of molecules are unable to cross the cell membrane; thus, the pH_cythomeostasis depends essentially on intracellular buffering power.     

Spatial aspects of intracellular pH regulation in heart muscle

Intracellular pH (pH_i) is an important modulator of cardiac function. Because it is readily influenced by metabolic processes, pH_i is controlled physiologically. Classical models of intracellular pH regulation comprise acid/base transport proteins expressed in the sarcolemma, acting in concert with intracellular buffers. These two processes are coupled via a diffusive movement of protons. Because intracellular H⁺ buffering is high, H_i⁺-diffusion occurs through a passive shuttling on intrinsic mobile buffers such as acetylated carnosine, anserine and homocarnosine: low molecular weight imidazole compounds. This mechanism is assisted by carbonic buffer, a system regulated biochemically by the enzyme carbonic anhydrase. H_i⁺-mobility via the buffer shuttles is low, and this can result in significant pH_i non-uniformity under conditions of high proton flux across the sarcolemma or within the cell. Spatial regulation of pH_i is complemented by passive H⁺ permeation between cells through gap junctions. This permeation is also mediated via protonated buffers. The control of pH_i is therefore dependent on carrier molecules that spatially shuttle protons within and between cells. In this review, we consider the physiological regulation of H_i⁺-mobility and permeation, and its relevance to pH_i-control in normal and pathophysiological states such as myocardial ischaemia, a clinical condition associated with severe intracellular acidosis.     

Identification of the interaction between the human recombinant histamine releasing factor/translationally controlled tumor protein and elongation factor-1 delta (also known as eElongation factor-1B beta)

The human recombinant histamine releasing factor (HrHRF), also known as translationally controlled tumor protein (TCTP), p23 and fortilin, has been described to have both extra- and intracellular functions. To elucidate an extra- or intracellular role for HrHRF, we used the yeast two-hybrid system with HrHRF as the bait and a Jurkat T cell library. We isolated a partial cDNA clone of the human elongation factor-1 delta (EF-1delta) encoding for amino acids 12 to 281. This interaction was confirmed by co-immunoprecipitation experiments. Previously, both HrHRF and EF-1delta have been isolated and identified in association with malignancy in numerous studies. EF-1delta is part of the EF-1 complex responsible for kinetic proofreading in protein synthesis. Additionally, DNA microarray data classifies TCTP (HrHRF) as co-regulated with ribosomal proteins and recent structural analysis of TCTP (HrHRF) relates it to a guanine nucleotide-free chaperone. Our findings of an interaction between HrHRF and EF-1delta taken with some of the recently published information concerning the TCTP (HrHRF) mentioned above suggest a possible intracellular role for TCTP/HrHRF.     

Interactions between fatty acids and alpha-synuclein

alpha-Synuclein (alphaS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, alphaS forms alpha-helices in the presence of negatively charged lipid surfaces. Moreover, alphaS was shown to interact with FAs in a manner that promotes protein aggregation. Here, we investigate whether alphaS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of alphaS signal in both (1)H and (13)C spectra and ii) the appearance of a very broad FA (13)C-carboxyl signal. These data exclude high-affinity binding of FA molecules to specific alphaS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were easily disrupted to form smaller particles in the presence of alphaS, indicating a direct protein-lipid interaction. We conclude that alphaS is not likely to act as an intracellular FA carrier. Binding to negatively charged membranes, however, appears to be an intrinsic property of alphaS that is most likely related to its physiological role(s) in the cell.     

PACE-1, a novel protein that interacts with the C-terminal domain of ezrin

The ERM proteins (ezrin, radixin, moesin) together with merlin comprise a subgroup of the band 4.1 superfamily. These proteins act as membrane cytoskeletal linker proteins mediating interactions between the cytoplasmic domains of transmembrane proteins and actin. To better understand how the ERM proteins function to regulate these junctional complexes, a yeast 2-hybrid screen was undertaken using ezrin as a bait. We describe here the identification and cloning of a novel protein, PACE-1, which binds to the C-terminal domain of ezrin. Characterization of PACE-1 in human breast cancer cell lines demonstrates it to have two distinct intracellular localizations. A proportion of the protein is associated with the cytoplasmic face of the Golgi apparatus. This distribution is dependent upon the presence of the PACE-1 N-terminal myristoylation consensus sequence but is not dependent on an association with ezrin. In contrast, PACE-1 colocalises with ezrin in the lamellipodia, where ezrin has a role in cell spreading and motility. A notable feature of PACE-1 is the presence of a putative N-terminal kinase domain; however, in biochemical assays PACE-1 was shown to have associated rather than intrinsic kinase activity. Together these data suggest that PACE-1 may play a role in regulating cell adhesion/migration complexes in migrating cells.     

Non-bicarbonate intracellular pH buffering of reptilian muscle

Summary Non-bicarbonate intracellular pH buffering values of skeletal and cardiac muscles were measured for 16 species of Australian reptiles from four orders (snakes, skelctal 19–36 slykes, cardiac 9–17 slykes; lizards, skeletal 25–54 slykes, cardiac 17–19 slykes; turtles, skeletal 25–43 slykes, cardiac 11–24 slykes; crocodile, skeletal 43 slykes). Although a positive correlation between pH buffering capacity and dependence on anaerobic muscle work was found, even the highest reptilian pH buffering values were low relative to equivalent white anaerobic muscles of fish, birds, and mammals. The low non-bicarbonate intracellular pH buffering capacity of reptilian muscle arises through lower contributions from proteins (10–14 slykes), non-protein histidine (7–18 slykes) and phosphate (5–15 slykes). It is concluded that while other vertebrates depend on these intracellular buffers for regulating muscle pH during anaerobic muscle work, reptiles rely less on buffering and instead may tolerate greater pH fluctuations.Abbreviations intracellular pH buffering capacity - EDTA ethylenediaminetetra-acetic acid - HPLC high performance liquid chromatography - I.D. internal diameter - LDH lactate dehydrogenase     

大分子拥挤对DNA结构的影响

大分子拥挤(macromolecular crowding effect)代表了细胞内高度拥挤状态,其源于非特异性容积排斥效应,是细胞内与pH、离子强度等同等重要的生理因素。生物大分子介导的拥挤环境对于DNA-DNA、DNA-蛋白质的相互作用以及DNA高级结构、细胞核或核区结构的稳定具有重要作用。在拥挤环境中,大分子总浓度的增加将增强溶质的浓缩倾向,从而降低溶液的自由能。拥挤效应是胞内大分子环境的总体反映,具有高度的缓冲性,保证了胞内反应的稳定进行及细胞功能的正常行使。     

Charge effects on folded and unfolded proteins

We develop a theory for the effects of charge on the stabilization of globular proteins. The folding process is modeled as occurring through a fictitious intermediate state along a two-part thermodynamic pathway in which the molecule (i) increases its density and then (ii) rearranges its ionic groups to the protein surface. The equilibrium for the binding of protons in salt solutions is assumed to be driven by the electrical potential due to the charge distribution, in addition to the intrinsic binding affinity and bulk proton concentration. The potential is calculated for inside and outside a porous sphere model of the protein using the Poisson-Boltzmann relation, wherein the interior dielectric constant is taken to be a linear function of the chain density. The model predicts the slope of the titration curves for native myoglobin in agreement with experiments by Breslow and Gurd (1962). From the similar experiments on the unfolded state, and from the experiments of Privalov et al. (1986) on the intrinsic viscosity of the unfolded molecules, the theory shows that the unfolded state has a much higher density than a chain in a theta solvent and that the density increases with ionic strength. In addition, from the free energy of proton binding to the protein, we also calculate the electrostatic contributions to protein stability, a major contribution deriving from changes in ionization. We consider the example of the stability of myoglobin as a function of pH, ionic strength, and ionic groups buried in the native protein structure. We show that although maximum stability of most proteins should occur at their isoelectric point, the burial of nontitratable groups should lead to maximum stabilities at pH values other than the isoelectric point.     

Acidification power: Indicator of metabolic activity and autolytic changes inSaccharomyces cerevisiae

Acidification power, defined as the sum of the spontaneous pH change determined after suspending yeast cells in water and the substrate-induced pH change after addition of glucose to the resulting suspension, reflects the level of cellular energy sources. Its use as an indicator of metabolic state of the cells was tested during a 120-h aerobic starvation. Its changes coincided with changes in cell viability, initial rate of endogenous oxygen consumption rate, cell ATP, extra- and intracellular buffering capacity, and the ability of cell-free extract to produce acidity by glucose fermentation. It was used as a sensitive marker of metabolic changes occurring during starvation, on treatment with glycolytic and respiratory inhibitors, and at elevated temperature.