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 Methods 7, 1983, 123-142) five-chamber gradient mixer has been replaced by a two-vessel device.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.     

On the pH-dependence of the light-induced hydrogen ion gradient in spinach chloroplasts

The dependence of the light-induced H⁺ gradient in chloroplasts (ΔpH) on external pH was examined using the distribution of aniline, an amine of low pK_a. ΔpH was essentially independent of pH over the range of 7–8. It was previously reported that ΔpH, determined from the distribution of relatively polar amines of high pK_a, decreased as the pH was lowered below 8. It is suggested that, in the case of amines of high pK_a, ΔpH values determined at low external pH values are too low because the permeability of chloroplasts to the amine cation relative to that of the unprotonated form may be significant.     

Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing

When hydrophobic samples, or membrane proteins, are disaggregated in buffers containing detergents (e.g. Nonidet P-40), urea and 2-mercaptoethanol, and applied at the cathodic end of a gel cylinder or slab for isoelectric separation, as routinely performed for two-dimensional techniques, a severe disturbance of the alkaline region of the pH gradient ensues. This phenomenon has been attributed to high protein loads, which supposedly overcome the buffering power of isoelectric carrier ampholytes. On the contrary, in the present study it has been found that this suppression of the alkaline end of the pH gradient is due to 2-mercaptoethanol, which is a buffer with pK 9.5. This compound ionizes at the basic gel end and is driven electrophoretically along the pH gradient, sweeping away, along its path, and focused carrier ampholytes.     

Dependence on pH of the activity of pig liver esterase

The dependence on pH of the kinetic parameters for the hydrolysis of phenyl acetate catalyzed by pig liver carboxylesterase was examined for purified high-isoelectric point and low-isoelectric point fractions of enzyme that were separated by isoelectric focusing. The values of k_cat are half-maximal at pH 4.3 and 5.1 for the high- and low-isoelectric point forms, respectively, and show a shallow dependence on pH with a value of n = 0.5. The absence of a change in the pH dependence of k_cat for the high-isoelectric point enzyme in the presence of high concentrations of methanol, which reacts with the acetyl-enzyme intermediate to give methyl acetate, provides evidence that the pH dependence is not caused by a change in rate-determining step. This means that if an imidazole group is involved in catalysis its pK must be perturbed downward by 2–3 units. The pH dependence of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ is biphasic with apparent pK values for dissociations of the free enzyme near 7 and 4 for both the high- and low-isoelectric point enzymes. Inhibition by a second molecule of substrate and by methanol are strongest for high-pH forms of the enzyme.     

The effect of pH and ionic strength on the electrophoretic separation of acidic glycosaminoglycans

Using the electrophoretical methods applied to this study it is possible to determinate the dissociation constants (pK) of acid glycosaminoglycans containing a carboxylic group. The pK-values of the six acid glycosaminoglycans separated from animal connective tissues determined in this work were: hyaluronic acid (HA), pK = 3.0; chondroitin sulfate A (CS-A), pK = 2.8; chondroitin sulfate C (CS-C), pK = 3.3; dermatan sulfate (CS-B), pK = 3.3; heparatin sulfate (HeS), pK = 3.1 and heparin (HeP), pK = 2.4 and were measured at a constant ionic strength of I = 0.164 (NaCl) and at 10 ± 2°C.Variation of ionic strength showed that physiological conditions seem to be most suitable for the electrophoretic separation of the glycosaminoglycans studied. A decrease of ionic strength causes increasing mobility but less accurate spots. In the case of increasing ionic strength the results are vice versa.The second spot for HA very often appeared when pH values higher than 2 were used for electrophoresis. The spot had the same form as the original, high intensity, but an undecided migration in the pH range near the pK value of HA (3.0).     

pH Variation of midpoint potential for three photosynthetic bacterial cytochromes c′. A link between physical and functional properties

Midpoint redox potential (E_M) versus pH curves are reported over the pH range 5 to 10 for the cytochromes c′ from three species of purple photosynthetic bacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris and Chromatium vinosum. In each case, theoretical curves are fitted to the data and pK values for the reduced (pH 5–5.5) and oxidized (pH 8–8.5) forms of the protein are found to influence the midpoint redox potentials. The oxidized form pK values in each case are found to correlate with previously determined pK values for variation in physical and/or spectroscopic properties. This correlation of functional and physical observables is discussed in terms of a possible mechanism of control of midpoint redox potential through heme iron-ligand bonding as moderated by the protein conformation in response to solution conditions. The reduced form pK values are discussed in terms of a mechanism which would alter the polarity of the heme environment, thereby influencing redox potentials.     

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.     

Reactions of the amino groups of RNAse A: III. The existence of pH-dependent values of pK

The rates of the trinitrophenylation of the amino groups of ribonuclease A (RNAse) with the specific reagent trinitrobenzene sulfonic acid have been studied at 27°C, between pH 7.0 and 9.9. From the variation of the velocity constants with pH it has been shown that the reaction is biphasic in the sense that for each amino group two pKs have been found: one (pK = 7.3–7.52) in the range of pH between 7.0 and 8.3 and the other (pK = 9.28–9.69) in the pH range 8.5–9.9. It is pointed out that when the experimental conditions approached one another, there was agreement between the pK values obtained from titrimetric and kinetic studies. Evidence is presented from the literature concerning the validity of the pK value near 7.5 for the ε-amino groups in RNAse. The studies were repeated with performic acid oxidized RNAse and the 10 ε-amino groups were found to be monophasic with pK values between 8.01 and 8.10. The α-amino group of the N-terminal lysine was biphasic with a pK of 7.26 (pH range 7–8) and 8.13 (pH range 8.2–9.5).     

Transmembrane pH gradients and functional heterogeneity in reconstituted vesicle systems

The pH-sensitive, membrane impermeant fluorescence probes 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine; pK_a = 7.2) and 1-naphthol-3,6-disulfonate (Naps pK_a = 8.2) can be simultaneously entrapped within the intravesicular aqueous compartment of unilamellar vesicles and reconstituted proteoliposomes, where they function as reliable reporters of the intravesicular pH. Because the two probes are sensitive to pH over different but overlapping ranges, the useful monitoring range for the co-trapped probe pair extends from pH 6.5 to 9. In vesicles pre-equilibrated at a given pH and then subjected to a sudden change in external pH, the rate and extent of the subsequent change in internal pH are identical at all times during the re-equilibration, regardless of which probe is used to monitor the change. However, in reconstituted bacteriorhodopsin proteoliposomes, the size of the transmembrane pH gradient generated in the light always appears greater when pyranine is used to monitor internal pH. This discrepancy can most readily be understood in terms of heterogeneity in the vesicle suspension, with at least two populations of vesicles, one active in proton and one inactive. A simple algorithm was developed which generates, from the observed internal pH changes for two probes of different pK_a, the percentage of vesicles which are inactive, as well as the actual internal pH of the active fraction. The applicability of this algorithm was subsequently confirmed using a suspension of vesicles in which the level of heterogeneity was deliberately altered by the addition of various amounts of gramicidin. The apparent transmembrane pH gradient for the vesicle population as a whole decreased with increasing gramicidin, as did the calculated percentage of vesicles able to maintain a pH gradient, while the transmembrane gradient calculated for the active vesicle fraction only was essentially unaffected by gramicidin.     

pH, urea and substrate gradients for the optimization of ultrathin polyacrylamide gel zymograms

The preparation of ultrathin polycrylamide gels with different kinds of gradients (pH, substrates, inhibitors) is described. By using these gels fro contact printing after isolectric with Ampholines or Immobilines and for diffusion tests, the influence of pH or increasing amounts of substrates or inhibitors on enzyme activities is studied. These methods are successfully applied for the optimization of zymogram techniques and for the easy characterization of industrial microbiol enzyme preparations for technological purposes. With buffer-generated pH gradient gels, the pH optimum of all isoenzyme activities is demonstrated by contatc printing; the total amount of esoenzyme acitivities dependent on pH is determined by a diffusion test. Gels with a linear gradient between 0 and 8 M urea are used for isoelectric focusing, diffusion tests and contact printing in order to differentiate the unfolding and denaturing effects of urea on isoenzymes. Alterations in polygalacturonase isoenzyme patterns dependent on urea concentration of denaturation but by the change of chargers. In respect to band sharpness and straightness urea can be added advantagenously up to 2 M without changing the isoelectric points or activities of the isoenzymes. for the reproducibility of zymograms it is interesting to see that different substrate concentrations reveal different isoenzyme patterns.     

The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

pK determinations via pH-mobility curves obtained by isoelectric focusing-electrophoresis: Theory and experimental verification

Basic equations have been derived linking the electrophoretic migration in a stationary pH gradient of simple, singly charged cations or anions and of mono- mono- valent ampholytes with the pKs of their ionizable groups. In the case of diprotic ampholytes, an equation and a curve are described calculating a correction factor to be applied to the mobility measurements, accounting for the influence of the opposite charge species on the mobility curve of the ion being measured. This correction factor is a function of ΔpK and increases exponentially with decreasing values of ΔpK. These theoretical considerations have been experimentally verified by running pH-mobility curves of colored compounds, such as methyl red, neutral red and dexorubicin. The pKs thus measured were in excellent agreement with the pKs obtained independently by spectrophotometric titrations.     

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.     

The pH dependence of the oxidation-reduction midpoint potential of cytochromes c2 in vivo

A recent report by Pettigrew et al. [Biochim. Biophys. Acta 430, (1976), 197–208] has examined the pH dependence of the oxidation-reduction midpoint potential of cytochromes c₂ in vitro. In media of low ionic strength, these workers identified several pKs on the oxidized forms of the cytochromes, and in some cases there were also pKs on the reduced species. In this work we examine the pH dependence of the midpoint potentials of the cytochromes in situ, attached to the chromatophore membrane. Under these conditions no pK values are detected, and we conclude that in vivo there is no net change in the protonation of cytochrome c₂ during oxidation or reduction.     

pH Dependence of the Efficiency of Binding of Iron Cations to the Donor Side of Photosystem II

Light-induced interaction of Fe(II) cations with the donor side of Mn-depleted photosystem II (PS II(–Mn)) results in the binding of iron cations and blocking of the high-affinity (HA_Z) Mn-binding site. The pH dependence of the blocking was measured using the diphenylcarbazide/2,6-dichlorophenolindophenol test. The curve of the pH dependence is bell-shaped with pK ₁ = 5.8 and pK ₂ = 8.0. The pH dependence of the O₂-evolution mediated by PS II membranes is also bellshaped (pK ₂ = 7.6). The pH dependence of the process of electron donation from exogenous donors in PS II(–Mn) was studied to determine the location of the alkaline pH sensitive site of the electron transport chain. The data of the study showed that the decrease in the iron cation binding efficiency at pH > 7.0 during blocking was determined by the donor side of the PS II(–Mn). Mössbauer spectroscopy revealed that incubation of PS II(–Mn) membranes in a buffer solution containing ⁵⁷Fe(II) + ⁵⁷Fe(III) was accompanied by binding only Fe(III) cations. The pH dependence of the nonspecific Fe(III) cation binding is also described by the same bell-shaped curve with pK ₂ = 8.1. The treatment of the PS II(–Mn) membranes with the histidine modifier diethylpyrocarbonate resulted in an increase in the iron binding strength at alkaline pH. It is suggested that blocking efficiency at alkaline pH is determined by competition between OH^– and histidine ligand for Fe(III). Because the high-affinity Mn-binding site contains no histidine residue, this fact can be regarded as evidence that histidine is located at another (other than high-affinity) Fe(III) binding site. In other words, this means that the blockage of the high-affinity Mn-binding site is determined by at least two iron cations. We assume that inactivation of oxygen-evolving complex and inhibition of photoactivation in the alkaline pH region are also determined by competition between OH^– and a histidine residue involved in coordination of manganese cation outside the high-affinity site.     

Stabilization of pH gradients in buffer electrofocusing on polyacrylamide gel.

pH gradients in buffer electrofocusing on polyacrylamide gel (BEF) have been stabilized for at least 100 hr, 25°C, by replacing the strongly acidic and basic anolyte and catholyte with isoelectric buffers identical to the terminal constituents of the pH gradient and gel. Such stabilization leads to a constant pI position of an electrofocused protein. No stabilization of pH gradients is achieved under otherwise identical conditions when strongly acidic and basic anolyte and catholyte are used.     

The proteoglycan region of the tumor-associated carbonic anhydrase isoform IX acts as anintrinsic buffer optimizing CO2 hydration at acidic pH values characteristic of solid tumors

The enzymatic activities of carbonic anhydrase (CA, EC 4.2.1.1) isozymes CA I, II, IX (catalytic domain (cdCA IX) and catalytic domain plus proteoglycan, flCA IX), XII and XIV were investigated as a function of pH for the CO₂ hydration to bicarbonate and a proton. The cytosolic isoforms CA I and II as well as the catalytic domain of CA IX, together with the transmembrane isoforms CA XII and XIV showed sigmoid pH dependencies of k_cat/K_M, with a pK_a of 6.90–7.10, showing thus optimal catalytic efficiency around pH 7. The full length CA IX had a similar shape of the pH dependency curve but with a pK_a of 6.49, having thus maximal catalytic activity at pH values around 6.5, typical of hypoxic solid tumors in which CA IX is overexpressed. The proteoglycan domain of CA IX (present only in this transmembrane isoform) may thus act as an intrinsic buffer promoting efficient CO₂ hydration at acidic pH values found in hypoxic tumors.     

Sodium-dependent activation of intestinal brush-border sucrase: Correlation with activation by deprotonation from pH 5 to 7

The activation of rabbit intestinal brush-border sucrase in the pH range 4.8 to 9.2 was studied as a function of sucrose concentration and temperature in a metal-free, n-butylamine universal buffer, both in the absence and in the presence of sodium. When sodium was absent, enzyme activation involved the simultaneous loss of two key protons (pK₁ of about 5.6), thus yielding a high-affinity, catalytically active enzyme conformation. Inactivation followed when a third key proton (pK₂ of about 8.4) was lost. When sodium was present, kinetic analysis in the pH range 4.8 to 7.2 revealed that sodium activation involves distinct effects on the two kinetic parameters, V_m and K_m. The V_m parameter seemed to conform to the classical rules of pH-dependent enzyme activation and implicated the release of a single proton whose apparent pK (pK_1y, about 5.6) was little affected by sodium. On the contrary, the K_m parameter was strongly influenced by sodium. Here, activation of rabbit sucrase seemed to involve release of a different proton whose apparent pK (pK_1x also of about 5.6 in the absence of sodium) was strongly shifted to more acid values by saturating sodium concentrations. The functional distinction between the above two protons explains the existence of strong affinity-type activating effects of sodium on rabbit sucrase, previously shown to be pH independent (F. Alvarado and A. Mahmood, 1979, J. Biol. Chem.254, 9534–9541).     

Properties of potato α-glucosidase

Potato tuber α-glucosidase has an isoelectric point of 4.7 and an apparent MW of 120 000. The enzyme has a neutral pH optimum (pH 6.5–7.0) and a K_m of 0.21 mM for p-nitrophenol-α-D-glucoside at pH 6.8 and 30°. Maltose and higher maltosaccharides are also substrates. The enzyme exhibits transglucosidase activity.     

Solubilization,stabilization and isoelectric focusing of human liver neuraminidase activity

Homogenate preparations of human liver have been prepared and over 75% of the particulate neuraminidase activity (which comprises approx. 90% of the total activity) has been solubilized using 0.85% (w/v) Triton X-100 in 25 mM phosphate buffer (pH 6.8). The solubilized neuraminidase activity is extremely labile, but can be stabilized for at least 4 weeks at 2–4°C, using 10 mM N-acetylneuraminic acid. Kinetic characterization of homogenate and solubilized supernatant fluid neuraminidase activities indicated comparable pH optimum curves (maximum activity at pH 4.5–4.7) and apparent K_m values (0.2–0.4 mM) for the synthetic fluorometric substrate $\text{4-}\text{methylbelliferyl}\text{-α-}\text{D}\text{-N-}\text{acetylneuraminic}$ acid. Isoelectric focusing has been performed on human liver homogenates and Triton X-100-solubilized neuraminidase activities, and the presence of several forms (4–6) with isoelectric points (pI values) between 4.4 and 5.2 has been demonstrated in both preparations. The similar kinetic and isoelectric focusing properties of the two preparations suggest that the solubilized enzyme activity is representative of the homogenate activity and that the solubilized enzyme is suitable for purification purposes.