Focusing of pepsin in strongly acidic immobilized pH gradients

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.     

Isoelectric focusing in immobilized pH gradients: generation and optimization of wide pH intervals with two-chamber mixers

A new technique for generating extended pH gradients (5 pH units) in Immobiline gels is reported. The previously described (J. Biochem. Biophys. Methods 7, 1983, 123-142) five-chamber gradient mixer has been replaced by a two-vessel device. A single mixture of the available Immobilines (pK 3.6, 4.6, 6.2, 7.0, 8.5 and 9.3) is made, with relative concentrations adjusted so as to produce the most uniform buffering power throughout the desired pH interval. This mixture is then divided into two portions, which are titrated to the extremes of the required pH span with an acidic titrant (Immobiline pK approximately 1) and a basic species (Immobiline pK 9.95). Highly reproducible pH gradients (pH 4-9) are thus generated, which appear extremely useful for the first dimensioned of 2-dimensional techniques. Our previously reported computer program has been implemented with an optimization algorithm which, given any cocktail of Immobilines, automatically adjusts the relative initial concentrations until the smoothest possible beta power is found. For the first time it is possible to perform IEF under controlled physico-chemical parameters: pH span and linearity, beta power, ionic strength and molarity of the buffering species.     

Ultrasonic absorption studies of protein-buffer interactions

The acoustic absorption of protein solutions in the presence of phosphate and other buffering ions has been studied in the physiological pH range. Buffers containing hydroxyl residues as titratable groups cause a pronounced increase of protein sound absorption, which is attributed to relaxation processes of proton transfer reactions between buffer ions and accessible imidazole and -amino groups of the protein surface. Amino group based buffers like Good's buffers do not induce additional sound absorption. Measurement of the ultrasonic absorption as a function of pH and of buffer concentration, and corresponding parameter fitting of the equation describing proton transfer relaxation processes has been used to evaluate equilibrium parameters. For the imidazole group of the amino acid histidine a pK value of 6.22 and for the imidazole group of the protein lysozyme a pK value of 5.71 have been determined. In hemoglobin the ligand-linked pK changes have been monitored by recording ultrasonic titration curves.     

Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry.

A theoretical development in the evaluation of proton linkage in protein binding reactions by isothermal titration calorimetry (ITC) is presented. For a system in which binding is linked to protonation of an ionizable group on a protein, we show that by performing experiments as a function of pH in buffers with varying ionization enthalpy, one can determine the pK(a)'s of the group responsible for the proton linkage in the free and the liganded states, the protonation enthalpy for this group in these states, as well as the intrinsic energetics for ligand binding (delta H(o), delta S(o), and delta C(p)). Determination of intrinsic energetics in this fashion allows for comparison with energetics calculated empirically from structural information. It is shown that in addition to variation of the ligand binding constant with pH, the observed binding enthalpy and heat capacity change can undergo extreme deviations from their intrinsic values, depending upon pH and buffer conditions.     

Proton flux measurements from tissues in buffered solution

Proton movement across plant cell membranes is part of many important physiological processes. The net proton flux to or from tissues can be determined non-invasively by measuring the proton electrochemical potential gradient in the adjacent solution. In buffered solution, some of the protons crossing the tissue boundary diffuse as proto-nated buffer whose flux is not included in the flux calculated from the proton (hydrogen ion) electrochemical gradient. In this theoretical paper, it is shown how experimenters can calculate the protonated buffer flux from the measured proton flux in solution. The ratio of these two components of total proton flux depends on the pH of the solution and on the concentration and pK of the buffer. For a given concentration of a buffer which has a single pK, the flux ratio rises with pH when the solution pH is lower than the buffer pK. The slope is about 2 on a log₁₀ scale. As the pH increases above the pK, the flux ratio levels off to approach its maximum. With mixed buffers, or one having two or more pK values, the flux ratios are additive: each buffer acts independently based on its concentration and its pK value. Unbuffered solutions always have the buffering effects of water itself and also of carbonates due to carbon dioxide dissolved from the atmosphere. In unbuffered solutions at pH 6, the flux carried by water and carbonate is about 1 % of the measured proton flux. This validates measurements of proton flux from tissues, made by a number of workers, in unbuffered solutions below pH 6.     

Synthesis of an hydrophilic, pK 8.05 buffer for isoelectric focusing in immobilized pH gradients

The synthesis of a new, pK 8.05 acrylamido weak base for isoelectric focusing in immobilized pH gradients (IPG) is here reported. This compound N,N-bis(2-hydroxyethyl)-N'-acryloyl-1,3-diaminopropane is strongly hydrophilic, and thus inhibits any potential hydrophobic interaction among proteins and the grafted basic groups in an IPG matrix. In addition, this novel buffer represents a step ahead towards the goal of closing the 'gap' between the commercially available Immobilines, pK 7.0 and 8.5. Owing to the large distance between these two neighboring pK values, it is difficult to arrange for linear narrow pH gradients in this region. IPG compositions obtained with this new buffer give highly linear pH gradients and protein profiles identical to those obtained with commercial Immobilines.     

Preparative isoelectric focusing in immobilized pH gradients. II. A case report

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.     

Acid-catalyzed hydration of reduced nicotinamide adenine dinucleotide and its analogues.

The rate of the primary acid modification reaction of 1,4-dihydronicotinamide adenine dinucleotide (NADH) and 1,4-dihydro-3-acetylpyridine adenine dinucleotide (APADH) and their analogues has been studied over a wide pH range (pH 1-7) with a variety of general acid catalysts. The rate depends on [H+] at moderate pH and becomes independent of [H+] at low pH. This behavior is attributed to substrate protonation at the carbonyl group (pK of NADH = 0.6). The reaction is general acid catalyzed; large solvent deuterium isotope effects are observed for the general acid and lyonium ion terms. Most buffers cause a linear rate increase with increasing buffer concentration, but certain buffers cause a hyperbolic rate increase. The nonlinear buffer effects are due to complexation of the buffer with the substrate, rather than to a change in rate-limiting step. The rate-limiting step is a proton transfer from the general acid species to the C5 position of the substrate. Anomerization is not a necessary first step in the case of the primary acid modification reaction of beta-NADH, in which beta to alpha anomerization takes place.     

Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids.

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions.     

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.     

Isoelectric focusing as the crow flies

The evolution of isoelectric focusing is traced back over the years, from a somewhat shaky origin to present-day immobilized pH gradients. Four generations of methodology are classified and discussed: (A) Kolin's approach, consisting of a two-step technique, generation of a pH gradient by diffusion followed by a rapid electrokinetic protein separation; (B) Svensson-Rilbe's approach, consisting of creating a pH gradient in an electric field by utilizing as buffers a multitude of carrier ampholytes, i.e. of amphoteric species possessing good buffering capacity and conductivity at their pI; (C) immobilized pH gradients, by which non-amphoteric buffers and titrants (acrylamido weak acids and bases), titrated around their pK values, are grafted (insolubilized) onto a polyacrylamide gel matrix and (D) mixed-bed carrier ampholyte-Immobiline gel, by which a soluble, carrier ampholyte generated pH gradient coexists in the same matrix with an insoluble, Immobiline generated, pH gradient.     

Isoelectric focusing in immobilized pH gradients: Principle,methodology and some applications

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described.Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacitu; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strenght.     

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.     

Roles of the beta 146 histidyl residue in the molecular basis of the Bohr effect of hemoglobin: a proton nuclear magnetic resonance study

Assessment of the roles of the carboxyl-terminal beta 146 histidyl residues in the alkaline Bohr effect in human normal adult hemoglobin by high-resolution proton nuclear magnetic resonance spectroscopy requires assignment of the resonances corresponding to these residues. Previous resonance assignments in low ionic strength buffers for the beta 146 histidyl residue in the carbonmonoxy form of hemoglobin have been controversial [see Ho and Russu (1987) Biochemistry 26, 6299-6305; and references therein]. By a careful spectroscopic study of human normal adult hemoglobin, enzymatically prepared des(His146 beta)-hemoglobin, and the mutant hemoglobins Cowtown (beta 146His----Leu) and York (beta 146His----Pro), we have resolved some of these conflicting results. By a close incremental variation of pH over a wide range in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer, a single resonance has been found to be consistently missing in the proton nuclear magnetic resonance spectra of these hemoglobin variants. The spectra of each of these variants show additional perturbations; therefore, the assignment has been confirmed by an incremental titration of buffer conditions to benchmark conditions, i.e., 0.2 M phosphate, where the assignment of this resonance is unambiguous. The strategy of incremental titration of buffer conditions also allows extension of this resonance assignment to spectra taken in 0.1 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane buffer. Participation of the beta 146 histidyl residues in the Bohr effect has been calculated from the pK values determined for the assigned resonances in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer. Our results indicate that the contribution of the beta 146 histidyl residues is 0.52 H+/hemoglobin tetramer at pH 7.6, markedly less than the 0.8 H+/hemoglobin tetramer estimated by study of the mutant hemoglobin Cowtown (beta 146His----Leu) by Shih and Perutz [(1987) J. Mol. Biol. 195, 419-422]. We have found that at least two histidyl residues in the carbonmonoxy form of this mutant have pK values that are perturbed, and we suggest that these pK differences may in part account for this discrepancy. Furthermore, summation of the positive contribution of the beta 146 histidyl residues and the negative contribution of the beta 2 histidyl residues to the maximum Bohr effect measured in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer suggests that additional sites in the hemoglobin molecule account for proton release upon ligation greater than the contribution of the beta 146 histidyl residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and 31P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies

With the aim to provide sensitive 31P NMR probes of intra- and extracellular pH gradients that may reach cellular acidic compartments in biological systems, new alpha-aminophosphonates were designed to meet basic requirements such as a low pK(a)s and a great chemical difference (Deltadelta(ab)) between the limiting 31P NMR chemical shifts in acidic (delta(a)) and basic (delta(b)) media. A series of six phosphorylated pyrrolidines and linear aminophosphonates were synthesized using aminophosphorylation reactions and were screened for cytotoxicity on cultured Müller cells. Among the compounds not being toxic under these conditions, three molecules were selected since they displayed the best in vitro (in several phosphate buffers and in a cytosol-like solution) properties as 31P NMR acidic pH markers, that is 3, 5 and 9, having the pK(a) values of 3.63, 5.89 and 5.66, respectively. The Deltadelta(ab) values of these pH markers were at least 3 times larger than that of standard 31P NMR probes, with a low sensitivity to ionic strength changes. From these data, it was proposed that 3, 5 and 9 could be used as reporting probes of subtle proton movements in acidic compartments, an area that still remains poorly investigated using non invasive 31P NMR methods.     

Maintenance of Acidic Lateral Intercellular Spaces by Endogenous Fixed Buffers in MDCK Cell Epithelium

The lateral intercellular spaces (LIS) of MDCK cell epithelia grown on permeable supports are about 0.4 pH units acidic to the bathing solutions, presumably because of buffering by the fixed negative charges on the surface of the lateral cell membranes. To test the hypothesis that fixed buffers are responsible for the acidity, a theoretical and experimental approach was developed for the determination of the concentration and pK of the fixed buffer constituted by the glycocalyx. The pH of the solution in the LIS was measured by ratiometric fluorescence microscopy while the buffer concentration or composition of the bathing solutions was altered. In addition, the divalent cation Sr²⁺ was added to the perfusion solutions to displace protons from the fixed buffer sites for the determination of the fixed buffer properties. We conclude that the LIS contain 3.7 mm of pK 6.2 fixed buffer and that this buffer is responsible for the acidic microenvironment in the LIS. Received: 9 April 1998/Revised: 28 July 1998     

Physico-chemical properties of amphoteric, isoelectric, macroreticulate buffers

We report here the properties of a new family of resins possessing an amphoteric character and able to strongly buffer at their pI values. They have been adopted as carriers for growth of cells in tissue culture and for hydroponics (Righetti et al. 1991; J. Biotechnol. 17, 169-176) but it is to be expected that such resins could have interesting chromatographic applications. It has been found that such beads [made by incorporating a pK 6.2 weak acrylamido base and a pK 4.6 weak acrylamido acid in a 2:1 ratio (thus with a pI of 6.2) into a neutral polyacrylamide backbone], independently from their initial conditioning (acid- or base-washed), spontaneously seek their equilibrium position (pI value) upon washing off excess titrant. Thus, upon potentiometric titration, they are seen to buffer in both directions of the pH scale (contrary to the behaviour of a pure carboxyl or a pure amino surface, which will exhibit only unidirectional buffering power). From the behaviour of these amphoteric beads when polymerized in the absence or in the presence of salts (0.2 M NaCl), it is hypothesized that, for exerting buffering power, both the buffering ion and its counterion must be incorporated non-randomly in the chain, but as a couple or in close proximity. Upon random incorporation of the two ions, buffering power is lost.     

pK-matched running buffers for gel electrophoresis.

Electrophoresis through agarose and polyacrylamide-type gels is the standard method to separate, identify, and purify nucleic acids. Properties of electrophoresis buffers such as pH, ionic strength, and composition affect performance. The buffers in use contain a weak acid or weak base buffered by a compound with a dissimilar pK. Herein, three pK-matched buffers were developed, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25 degrees C (within 0.3 pK units): (i) Ethanolamine/Capso, pH 9.6; (ii) triethanolamine/Tricine, pH 7.9; and (iii) Bis-Tris/Aces, pH 6.7. On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/acetate, pH 8.0) or TBE (Tris/borate, pH 8.3) buffers. The buffers were also tested in 7 M urea denaturing LongRanger sequencing gels and in nondenaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions (up to 200x), and lower current per unit voltage, resulting in less heat generation.     

alpha - and beta -phosphorylated amines and pyrrolidines, a new class of low toxic highly sensitive 31P NMR pH indicators. Modeling of pKa and chemical shift values as a function of substituents

Fourteen linear and cyclic alpha- and beta-aminophosphonates in which the P-atom is substituted by alkoxy groups have been synthesized and evaluated as (31)P NMR pH markers in Krebs-Henseleit buffer. pK(a) values varied with substitution in the range 1.3-9.1, giving potentially access to a wide range of pH. Temperature had a weak influence on pH and a dramatic increase in ionic strength slightly modified the pK(a) of the pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH).All compounds displayed a 4-fold better NMR sensitivity than inorganic phosphate or other commonly used phosphonates, as assessed by differences delta(b)-delta(a) between the chemical shifts of the protonated and the unprotonated forms. In isolated perfused rat hearts, a non-toxic concentration window of 1.5-15 mm was determined for three representative compounds. Using empirical linear relationships, the experimental values of pK(a), delta(a), and delta(b) have been correlated with the two-dimensional structure, i.e. the chemical nature of substituents bonded to the secondary amine and P-atom. The data suggest that DEPMPH and its cyclic and linear variants are ideal versatile (31)P NMR probes for the study of tenuous pH changes in biological processes.     

Two-dimensional maps in very acidic immobilized pH gradients

Up to the present time it has been impossible to perform two-dimensional (2-D) separations in very acidic immobilized pH gradients (IPG), due to the lack of suitable buffering acrylamido derivatives to be incorporated into the polyacrylamide matrix. The advent of the pK 3.1 buffer (2-acrylamido glycolic acid; Righetti et al., J. Biochem. Biophys. Methods 16, 1988, 185–192) allowed the formulation of such acidic gradients. We report here separations in IPG pH 2.8–5.0 intervals of polypeptide chains from total lysates of rat intestinal and liver cells and 30S and 50S ribosomal proteins from Halobacterium marismortui. Conditions are given for highly reproducible first and second dimensions gels and for a proper silver staining of 2-D maps with practically no background deposition.