Search

High inter- and intra-individual variability in the pH of fluids in the human gastrointestinal (GI) tract has been described in the literature. The aim of this study was to assess the influence of physiological variability in fasted pH profiles of media along the GI tract on diclofenac sodium (DF-Na) dissolution from matrix tablets. Four individual in vivo fasted pH profiles were selected from the literature that differed in pH values and transit times from the stomach to the proximal colon. Using a glass-bead device flow-through dissolution system, these pH profiles were simulated in vitro using a specific media sequence and considering simulated intestinal buffer capacities corresponding to in vivo literature data. Dissolution experiments were then performed in the same system with media sequence following individual pH profiles. In dissolution experiments, where influences of simulated gastric emptying time (GET), gastric pH value, small intestinal transit time, and colonic pH were studied; high influence of gastric pH value and GET on DF-Na dissolution was observed. The effect of variability in pH profiles in the range of individual in vivo data on DF-Na dissolution was also clearly observed in experiments, where dissolution studies were performed following three simulated in vivo individual pH profiles. The differences in DF-Na release between three individual pH profiles were substantial; they also reflected in simulated plasma concentration profiles and can be attributed to pH dependent diclofenac solubility.

Hydrogel beads are particularly promising vehicles for the encapsulation, protection, and release of bioactive agents in foods, supplements, and pharmaceuticals. However, most hydrogel beads are highly porous so that their internal pH is typically fairly similar to that of the surrounding solution, which can lead to degradation of encapsulated pH-sensitive bioactives. In this study, hydrogel beads were fabricated with self-regulating internal pH microclimates by encapsulating Mg(OH)2 inside them. This buffer is insoluble at neutral pH, but dissolves under acidic conditions thereby releasing OH− ions that neutralize H+ ions. A quantitative fluorescence confocal laser scanning microscopy (CLSM) method was developed to map the local pH inside the hydrogel beads, which was based on trapping a pH-sensitive dye (fluorescein tetramethylrhodamine dextran, FRD) inside the beads. Hydrogel beads were prepared by injecting a solution containing alginate (gelling agent), Mg(OH)2 (buffer) and FRD (pH probe) into another solution containing calcium chloride (cross-linking agent) using a commercial encapsulation unit. The fluorescence CLSM method was then used to detect the pH profile inside the beads after they were exposed to highly acidic simulated gastric fluids (SGF). Before exposure to SGF, the pH profile inside buffer-free beads was fairly uniform (pH 6.8–7.0), whereas the pH at the center of the hydrogel beads was slightly higher than that at the edge for Mg(OH)2-loaded beads (pH 7.6–7.2). After exposure to SGF, the pH inside buffer-free beads rapidly became highly acidic (around pH 3), whereas the pH inside Mg(OH)2-loaded beads remained fairly similar to the initial value (around pH 7.5). The CLSM method developed in this work may be useful for quantifying the local pH in other systems, while the buffer-loaded hydrogel beads may be useful for encapsulation of acid-labile bioactives.

Recently, dynamic approach has been applied to determine the steady state concentrations of multiple ionic species present in complex buffers at equilibrium. Here, we have used the dynamic approach to explicitly model the pH profiles of biologically relevant phosphate buffer and universal buffer (a mixture of three tri-protic acids such as citric acid, boric acid and phosphoric acid). The results from dynamic approach are identical to that of the conventional algebraic approach, but with an added advantage that the dynamic approach, allow for the modelling of complex buffer systems relatively easy compared to that of algebraic method., Graphical abstract fx1, Highlights • Accurate model proposed for Phosphate and Universal buffer using dynamic approach. • Optimization of pKa values in phosphate and universal buffer using dynamic approach. • A method that than be easily extended to predict pH of complex bio-fluids if concentration of metabolites and its pKa values are known.

This study was conducted to determine an effective completion point of the simultaneous removal of NH 4 + –N and Mn 2+ , using a biological aerated filter (BAF). The simultaneous removal was performed and monitored under two operation modes: i.e., batch and continuous. Each mode was operated by supplying continuous aeration for 7.5 h and intermittent aeration for 6 h. The results showed a higher performance of the simultaneous removal of NH 4 + –N and Mn 2+ for the batch with full operation of aeration. The effective curves were detected for dissolved oxygen (DO), oxidation-reduction potential (ORP) and pH profiles, correlating with the complete simultaneous removal of NH 4 + –N and Mn 2+ . By recognising the curves, the complete simultaneous NH 4 + –N and Mn 2+ removal can be predicted effectively, affecting a reduction in human capital and operating costs.

Common drugs intended for action in plasma (antibacterials, antiallergics, diuretics...) often display both acidic and basic behavior, and some of these amphoteric compounds can appear as zwitterions. In such cases, accurate profiling of lipophilicity vs. pH, which plays a fundamental role in drug pharmacokinetics, might be complex. In the present work two common lipophilicity determination methods based on the drug distribution between 1-octanol and aqueous buffer i.e. phase equilibration (shake-flask) and two-phase titration (potentiometry), were compared with a high-throughput lipophilicity index, the Chromatographic Hydrophobicity Index (CHI). The results were also compared with log Do/w pH-profiles calculated by different algorithms from ACD/Labs. Accurate and similar results were obtained for both octanol-water approaches but, due to the lower determination times and the absence of different ion-pairing buffers, potentiometry was shown to be the most convenient method. CHI vs. pH profiles provide rapid and efficient information, which is very convenient for lipophilicity screening purposes, but may differ slightly from shake-flask and potentiometric results.

To investigate the permeation of two ionisable drug molecules, warfarin and verapamil, across artificial membranes. For the first time since the introduction of the parallel artificial membrane permeation assay (PAMPA) in 1998, in situ permeation-time profiles of drug molecules are studied.The method employs a rotating-diffusion cell where the donor and acceptor compartments are separated by a lipid-impregnated artificial membrane. The permeation of the solute is investigated under well-defined hydrodynamic conditions with control over the unstirred water layer. The flux of the permeating molecule is analysed in situ using UV spectrophotometry.In situ permeation-time profiles are obtained under hydrodynamic control and used to determine permeability coefficients. An advanced analytical transport model is derived to account for the membrane retention, two-way flux and pH gradient between the two compartments. Moreover, a numerical permeation model was developed to rationalise the time-dependent permeation profiles. The membrane permeability, intrinsic permeability and unstirred water permeability coefficients of two drug molecules are obtained from two independent methods, hydrodynamic extrapolation and pH profiling, and the results are compared.Both warfarin and verapamil exhibit high permeability values, which is consistent with the high fraction absorbed in human. Our results demonstrate that a considerable lag-time, varying with the solute lipophilicity and stirring rate, exists in membrane permeation and leads to incorrect compound ranking if it is not treated properly. Comparison of the permeability data as a function of pH and stirring rate suggests that some transport of the ionized molecules occurs, most likely via ion-pairing.

The objective of this study was to compare a novel sustained release tablet formulation that has the potential to be used for drugs of different physicochemical properties using a binary mixture of polymethacrylate polymers in their salt forms with the polymethacrylate interpolyelectrolyte complex (IPEC) tablets in terms of drug release and compactness. Also, we aimed to compare this formulation with an IPEC tablet in terms of drug release. Tablets prepared using Eudragit E-Citrate and Eudragit L-Sodium were more convenient, easier to prepare, and showed better sustained release and compactness characteristics compared to IPEC tablets of similar concentrations and preparation methods.

The Chromatographic Hydrophobicity Index (CHI) has been measured for several diprotic compounds. In order to model the variation of CHI with pH, a general equation for polyprotic compounds which allows the calculation of CHI for each one of the species has been proposed. Different types of diprotic solutes have been selected: neutral acids, neutral bases, and amphiprotic compounds (non-zwitterionic and zwitterionic). The applicability of the model to these complex polyprotic compounds, its advantages and limitations, as well as the information provided by the fits is discussed. In general, the model fits the experimental behaviour of all kind of solutes very well, obtaining the higher hydrophobicity values for the neutral form of the compounds, except for the zwitterionic phenylalanine. The equation and parameters obtained allow an easy calculation of CHI of the compound at any pH value.

Numerical simulations are presented for ampholyte-based isoelectric focusing in 2D microgeometries. In this study, model proteins are focused in the presence of 25 biprotic ampholytes under an applied electric field. Each protein is considered as a simple polypeptide having ten charge states, while the biprotic ampholytes are selected to generate a shallow pH range of 6 to 9. Straight and contraction-expansion microchannels are considered here, and a nominal electric field of 300 V/cm is maintained for separation of proteins. Six distinct values of ΔpKs between 1 and 3.5 are investigated for ampholytes to form pH profiles in a 1 cm long microchannel. Simulation results show that relatively larger values of ΔpK (ΔpK > 3 are required to form stepless pH profiles in the system. The peak heights and differential resolutions of focused proteins are much higher for lower values of ΔpK for which a stepped pH profile is evident. For each protein, the time it takes for the two edges of a peak to merge increases linearly with ΔpK, while the focusing time goes up exponentially with increasing ΔpK. Both merging and focusing times of protein are higher for contraction-expansion microchannel than those of straight microchannel. For a particular value of ΔpK, the contracted "Zoom" region of contraction-expansion channel is able to form more tightly focused bands than the expanded region.

This paper discusses Excel applications related to the prediction of drug absorbability from physicochemical constants. PHDISSOC provides a generalized model for pH profiles of electrolytic dissociation, water solubility, and partition coefficient. SKMODEL predicts drug absorbability, based on a log-log plot of water solubility and O/W partitioning; augmented by additional features such as electrolytic dissociation, melting point, and the dose administered. GIABS presents a mechanistic model of g.i. drug absorption. BIODATCO presents a database compiling relevant drug data to be used for quantitative predictions.

Catalase was immobilized on alumina carrier and crosslinked with glutaraldehyde. Storing stability, temperature and pH profiles of enzyme activity were studied in a column reactor with recirculation and in a batch stirred-tank reactor. The immobilized enzyme retained 44% of its activity at pH 11, 30 ◦ C and 90% at 80 ◦ C, pH 7. The half-life time of the immobilized catalase was increased to 2 h at pH 12, and 60 ◦ C. Acceptable results were achieved when the residual water from the washing process of H2O2-bleached fabrics was treated with the immobilized enzyme and then reused for dyeing.

Process dynamics in Anammox systems were evaluated through continuous monitoring of pH, oxidation reduction potential (ORP) and conductivity in two separate newly started-up sequencing batch reactors, one seeded with an enriched Anammox sludge and the other seeded with mixed activated sludge. The pH and ORP profiles exhibited characteristic patterns depending on the process dynamics during early start-up, start-up and enrichment phases of the operational period of 410 days. The simultaneously continuing processes of the start-up period showed apparent indicative trend lines in pH and ORP profiles. Conductivity profiles were consistent with the process dynamics in all phases. During the enrichment phase, conductivity decreases could quantitatively be related to process removal efficiencies and all real-time profiles exhibited specific break-points which coincided with the end of Anammox in each cycle. The end of Anammox was observed as an ‘apex’ on pH profiles and a ‘valley’ on ORP profiles. The ‘apex’ and ‘valley’ points exactly coincided with the end point of the linear decrease in the conductivity profiles. The overall findings suggested a great potential in using real-time pH, ORP and conductivity measurements for quick and reliable monitoring of Anammox systems during start-up and enrichment periods.

A new pH selective microelectrode has been developed and tested. The described microelectrode is based on the neutral chromoinophore ETHT 2418 which is incorporated into a liquid membrane of variable composition. The microelectrode is based on an optimized membrane composition and exhibits a linear dynamic response function from pH

The pH control was important for curdlan production with Agrobacterium sp. ATCC31750. Specific cell growth rate was the highest at pH 7 and the specific curdlan production rate was at pH 5.5. The pH profiles maximizing curdlan production was changed from pH 7 optimal for cell growth to pH 5.5 optimal for curdlan production after ammonium consumption. The feedback inferential control methods, with easily measurable variables such as NaOH addition for pH control and dissolved oxygen (DO), were also applied. The pH was successfully controlled to follow optimal profiles and the maximal production of curdlan (60 g l−1 in 120 h) was achieved with feedback optimal control.

In order to gain some insights into durum wheat lipoxygenase (LOX) biochemistry and in particular in order to find out whether the antioxidants α-tocopherol and L-ascorbate can prevent the LOX-dependent semolina bleaching, which leads to a low quality of pasta product, a study of LOX activity in a crude protein extract from semolina was performed by means of spectrophotometric techniques. Measurements made of linoleate hydroperoxidation reaction (λ=234 nm) and of the coupled β-carotene bleaching reaction (λ=460 nm), demonstrated the existence of more than one enzyme exhibiting LOX activity as revealed by the different pH profiles. The linoleate hydroperoxidation reaction exhibited a sigmoidal dependence on the substrate concentration (K0·5=60±0·67 μ M ,Vmax =1·12±0·055 units/g of semolina), while β-carotene bleaching showed a hyperbolic relationship and followed the Michaelis-Menten equation (Km=3·3±0·53 μ M , Vm=0·030±0·0062 units/g). Both linoleate hydroperoxidation and β-carotene bleaching were strongly inhibited by added α-tocopherol. In both cases, the enzyme affinity for the substrate was reduced, while the Vmaxwas not affected. In particular, β-carotene bleaching was inhibited in a competitive manner (Ki=7·7±0·21 μ M ). L-ascorbate also inhibited both reactions: the Vmaxof linoleate hydroperoxidation was lowered, while theK0·5 remained unchanged; in the case of β-carotene bleaching the inhibition was uncompetitive (Ki=445±71·5 μ M ). Therefore, the use of both α-tocopherol and L-ascorbate to reduce semolina bleaching during pasta making is suggested. A purification procedure of semolina LOX is also reported, showing the existence of two isoforms.

2-Hydroxypropyl-β-cyclodextrin (2HP-β-CyD) and 2,6-di-O-methyl-β-cyclodextrin (DIMEB) were tested as solubilizing agents for dihydroergotamine mesylate, with the aim of improving the pharmacotechnical properties of this drug. The pH dependence of the complexation was investigated on the basis of solubility/pH profiles. For uncomplexed dihydroergotamine mesylate, the solubility increased sharply in the acidic range. The same pH dependence was observed for 2HP-β-CyD and DIMEB. The shape of the pH profiles was not altered, but the pH profiles occurred at considerably higher concentrations than for dihydroergotamine mesylate alone. The solubilizing effect clearly increased in the acidic range, and the effects were stronger for DIMEB than for 2HP-β-CyD. The maximum stability of dihydroergotamine mesylate was shown to be pH 4.0. The degradation rates of dihydroergotamine mesylate in β-CyD solutions are not very different from those in pure dihydroergotamine mesylate solution. DIMEB has the most favourable effect on the stability, while 2HP-β-CyD causes higher degradation. During autoclaving, however, dihydroergotamine mesylate was to some extent protected against degradation by 2HP-β-CyD complexation. Nevertheless degradation was so strong that autoclaving is not possible.

Catalytic reactions in the unstirred layers near bilayer lipid membranes can induce nonmonotonic concentration profiles near the membrane surface. In the case of transmembrane diffusion of a substrate immediately followed by its conversion due to the presence of an aqueous soluble enzyme the size of the unstirred layer defined in terms of the concentration gradient at the membrane surface does not correspond to the width of the! aqueous layer adjacent to the membrane where the concentration differs from the bulk phase concentration. Deducing of flux values or convection parameters from the concentration gradient at zero distance from the membrane gives misleading results. An empirical equation for the estimation of the size of the concentration boundary layer is proposed. It was derived from pH profiles registered with the help of a microelectrode near a planar bilayer lipid membrane surrounded by a buffer solution containing at one side of the membrane aa;taldehyde and sodium acetate and at the other side alcohol dehydrogenase. Since this parameter equals to the thickness of the unstirred, layer in the case of exponential concentration profiles it may be applied to estimate both mass transfer restrictions and kinetic of diffusion limited reactions occurring in the immediate membrane vicinity regardless the complexity of the system under investigation.

Bidirectional isotachophoretic migration was confirmed by the direct measurement of the pH profiles and the boundary velocities of the separated zones. The anolyte used was 10 mM HCl—β-alanine (pH 3.6) and the catholyte was 10 mM KOH-acetic acid (pH 4.8). It was shown that bidirectional isotachophoresis could be achieved with an electrolyte system consisting of a leading electrolyte for an anionic analysis and one for a cationic analysis. The combination was not arbitrary but the pH difference between the anolyte and the catholyte was restricted to keep the effective mobility of the terminating ion not too small. The simultaneous separation and determination of anions and cations in a test mixture were demonstrated on the basis of the time-based zone length measured by the use of a dual detection system.

Routine cell culture is done in small-scale disposable vessels (typically 0.1-100 mL volumes) in academia and industry. Despite their wide use in bioprocess development (i.e., process optimization and process validation), miniature process scouting devices (PSDs) are considered "black boxes" because they are generally not equipped with sensors. In this study, we show that on-line monitoring of dissolved oxygen (DO) and pH in a T-75 flask-based PSD can be achieved during cell passaging and that this information can be linked to different cellular metabolic states. In this case, on-line monitoring of DO and pH show three distinctive metabolic regions in passages 1-18, 19-28, 29-54 and in particular, the shift in the pH curve, the specific oxygen uptake rate (q(O2)), and the lactate production rate to the oxygen consumption rate yield (Y(Lac/ox)) confirm the existence of these distinctive metabolic regions. These findings are particularly useful because they show that sensor equipped PSDs can help to monitor cell culture behavior after thaw, in pre- and seed culture prior to scale-up and in development/optimization studies. Such routine monitoring will help to develop more consistent cell culture techniques.

A method for optimization of the mobile phase composition in ion exchange chromatography with a wide range external pH gradient (approx. over 5 pH units) has been formulated. It is shown that programming of various wide range pH profiles which are not impaired by buffer interactions of the mobile phase with sorbent is possible. Utilization of the wide range external pH gradient in cation-exchange chromatography of bovine haemoglobin is also demonstrated.

Primary and secondary inner filter effects were employed to enhance the sensitivity of fluorescence measurements by using multiple indicator systems with complementary pH profiles. pH-sensitive absorbing indicators were mixed with pH-sensitive fluorescent indicators possessing complementary spectral overlap and pK a 's. The resulting solutions possessed greater fluorescence pH sensitivity. The pH changes were followed by monitoring the fluorescence emitted by one of the indicators

The most often used methods for the measurement of intragastric acidity are repeated mostly hourly aspirations of gastric contents or continuous pH measuring with an electrode. Intragastric pH, volume of aspirated gastric juice and total acid secretion have been tested over relatively short periods of time (2-12 h) and through an entire 24-hour period. Nearly every clinical antiulcer drug which neutralizes acidity or inhibits acid secretion has been investigated with this method. pH monitoring with the gastric aspiration procedure is a reliable and reproducible method. There is a remarkable global agreement in the results. While pH electrodes provide an alternative means of measuring pH, they have the disadvantage of not allowing measurement of volume and/or acid secretion.

The two commercially available Immobilines having a pK of 6.2 (2-morpholino ethyl acrylamide) and 7.0 (3-morpholinopropylacrylamide) have been modified and two new buffers have been synthesized: 2-thiomorpholinoethylacrylamide, pK 6.6, and 3-thiomorpholinopropyl acrylamide, pK 7.4. The replacement of an oxygen with a sulfur atom in the morpholino ring is thus seen to shift the pK values of these two bases by +0.4 pH units. In formulations in which the two new bases replaced the standard morpholino derivatives, identical pH profiles and protein patterns were obtained. The reason for this work was to try to close the gap between the pK 7.0 and 8.5 species and to provide the users of immobilized pH gradients with more buffers in the neutral pH region. The two new thiomorpholino derivatives are an important step in this direction.

While one-dimensional SDS-PAGE separates proteins on the basis of size, two-dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two-dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations. In addition there are support protocols for determining pH profiles of gels, casting Immobiline gels, preparing cell and tissue samples for isoelectric focusing, preparing molecular weight standards, and using two-dimensional protein databases.

The separation of dilute protein mixtures was achieved using simple monovalent buffering species to form retained, internally produced pH gradients on a strong-basic anion-exchange column. Highly focused proteins bands localized on stepwise pH transitions were produced experimentally under trace and volume overloaded feed conditions. Numerical simulations were performed that accurately predict the pH profile and protein band shapes in the column effluent. Experimental results were combined with numerical investigations to explore strategies for designing efficient preparative-scale chromatofocusing systems using simple, inexpensive buffers and adsorbents.

Rat liver microsomes and cytosol have been co-immobilized by microencapsulation into artificial cells. The immobilized enzyme retained good enzyme activity. Enzyme kinetics, storage stability, pH profiles and temperature profiles have been analyzed.

The electrophoretic mobility of commercial alumina and titania powders and their mixtures (ratio of alumina:titania mixtures (by weight) 1:2 and 4:1) dispersed in 10 −3 mol dm −3 KNO 3 was determined as a function of pH in the range pH 3–11, using both microelectrophoresis and mass transport methods. The results are in good agreement with the previous experimental results. The value of the isoelectric point (IEP) for alumina and titania was found to be at pH9 and 5.5, respectively. It appears that the dispersion concentration has no significant effect on the electrophoretic mobility versus pH. The results also suggest that the addition of titania to alumina has lowered the IEP value from pH ∼9 to ∼8.4 and the addition of alumina to titania has increased the IEP value from pH ∼5.5 to 6. The electrophoretic mobility versus pH profiles for the mixtures of alumina and titania powders appear to represent the electrokinetic behavior of both alumina and titania powders; however, the shape of the electrophoretic mobility versus pH profiles and the position of the isoelectric point appear to be governed by the powder with a large total surface area in the mixture, but not necessarily the weight.

SummaryDialysing milk against phosphate-free sera showed that the transition in the ethanol (EtOH) stability/pH profile was associated with the soluble phosphate component of the milk serum. Sigmoidal behaviour similar to that of milk was reproduced when the EtOH stability of artificial mixtures of casein, Ca and phosphate was measured as a function of pH. A mechanism for the s coagulation of skim-milk is discussed.

The degradation of mitomycin C in acidic medium has been investigated utilizing a stability indicating high-performance liquid Chromatographic assay and ultraviolet spectrophotometry. Rate constants for the decomposition at 20°C are determined and a pH profile was constructed. The ratio between the two mitosene degradation products as function of pH is quantified. The degradation mechanism of mitomycin C in acidic solution is evaluated.

Addition of small amphoteric substances or separators to the carrier ampholytes used in isoelectric focusing improves the separation of protein components. The technique has been applied to partially oxidized human hemoglobin, goat anti-bovine pancreatic oxidized ribonuclease, human γ-globulin, and barley β-amylase. Each of these proteins is a complex mixture of species which can be resolved selectively and reproducibly by this method. Superior separation is provided by this technique when compared to focusing in commercially available narrow-range ampholytes. Addition of separators usually results in the flattening of the pH profile of the gel. Inspection of the protein patterns indicates that no new protein bands arise, although different patterns of existing components are obtained by choice and concentration of separator as well as by concentration of carrier ampholytes. This technique, thus, offers the advantage of making conventional isoelectric focusing more versatile.

3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g−1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent Km values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced.

It is possible to measure pH values in ultra-narrow immobilized pH gradients (IPGs) when the polyacrylamide matrix contains a secondary, carrier ampholyte generated pH gradient. After an IPG run, the buffering ampholytes, contained in 5 mm gel segments, along the focusing axis, are eluted and the following measurements are made: pH, conductivity, buffering capacity and concentration profiles of carrier ampholytes. Local concentration maxima of buffering ampholytes correspond to maximum conductivity and buffering capacity. The same relationship applies to local minima of the same parameters. No single commercial carrier ampholyte mixture ensures smooth conductivity and buffering capacity profiles and even pH gradients over very narrow pH intervals (0.3 pH unit spans). The best profiles are obtained only with mixtures of different commercial carrier ampholytes. Across neutrality, ca. 10% total buffering ampholytes should be added to an ultra-narrow IPG gradient to ensure an average buffering power (β av. ) of ca. 3 mequiv./1 · pH and an average conductivity (λ av. ) of 20–30 μS, while in acidic (around pH 4 and below) and alkaline (above pH 9) pH intervals only one third of this concentration is needed, as the carrier ampholytes exhibit substantially higher λ and β values and the hydrolytic products of water begin to contribute to the buffering capacity and conductivity of the system.

Purified glucose isomerase from Actinoplanes missouriensis was immobilized on porous DEAE-cellulose beads by simple adsorption. The immobilized glucose isomerase retained over 70% of its original activity. The hinderance of immobilized enzyme activity due to pore diffusion and film diffusion was insignificant with the bead size at 35 mesh or smaller. The relative substrate flow rate can be kept at 0.04 cm/s or higher. The optimum pH of the imobilized enzyme did not change, however, the optimum pH range became broader. The broader pH profile indicated that immobilized enzymes are less sensitive to pH change in the substrate. The half life of the immobilized enzyme was at around 1,000 h at 60°C. Cobalt ions are not required for enzyme stability. The cost of using immobilized enzyme on DEAE cellulose beads should be less be than that of the whole cell immobilization system due primarily to the fact that DEAE-cellulose beads are reusable for immobilization as well as for enzyme purification. Auf DEAE-Celluloseperten immobilisierte Glucoseisomerase Gereinigte Glucoseisomerase aus Actinoplanes missouriensis, wurde durch einfache Adsorption auf porosen DEAE-Celluloseperlen immobilisiert. Die immobilisierte Glucoseisomerase behielt uber 70% ihrer ursprunglichen Aktivitat. Die Behinderung der immobili-sierten Enzymaktivitat durch Poren- und Filmdiffusion war bei einem Perlendurchmesser von 35 Maschen oder geringer unbedeutend. Die relative Fliesgeschwindigkeit kann bei 0,04 cm/s oder hoher gehalten werden. Das optimale pH des immobilisierten Enzyms anderte sich nicht, aber der optimale pH-Bereich wurde breiter. Das breitere pH-Profil deutete darauf hin, das immobilisierte Enzyme gegenuber pH-Anderungen weniger empfindlich sind. Die Halbwertzeit des immobilisierten Enzyms betrug bei 60°C etwa 1 000 h. Kobaltionen sind fur die Stabilitat des Enzyms nicht erforderlich. Die Kosten fur die Verwendung von auf DEAE-Celluloseperlen immobilisiertem Enzym sollten niedriger sein als bei dem vollstandigen Zellimmobilisierungssystem, wenn man berucksichtigt, das DEAE-Celluloseperlen sowohl fur die Immobilisierung als auch fur die Reinigung des Enzyms wiederverwendet werden konnen.

o-Diphenolase extracted from Triticum aestivum (cv. Nettuno) and purified in various steps gave a 250-fold purification over the crude extract. This purified enzyme showed maximum relative activity towards 4-methylcatechol, generally high or moderate activity towards di- and polyphenols, and very low activity towards monophenols. Total activity, determined at each step of purification, showed that treatment with ammonium sulphate and calcium phosphate gel gave rise to an activation of o-diphenolase, thus revealing a latency of enzymatic activity. In the pH profile activity towards 4-methylcatechol two optima at pH 5.3 and 6.9 were observed.

A pH-rate method, based on following the time dependence of hydroxide release by monitoring the pH drift as a funetion of time, is developed and applied to the construction of a detailed log activity vs pH profile for yeast pyruvate decarboxylase. In the pH range of 5 to 7 at 30°C this method reproduces the results of conventional initial rate studies but, in addition, produces a hitherto unreported pH independent activity region between pH 5.4–5.8. At least four pKs can be discerned in the pH-activity profile of this enzyme, instead of the two previously reported.

1. 1. This study was undertaken to characterize the α-glucosidase present in blood and seminal plasma of two strains of pure-bred rams which are known as seasonal breeders. 2. 2. pH profiles and activity levels were investigated in blood and seminal plasma using a sensitive spectrophotometric assay with para-nitrophenyl-α- d -glucoside as substrate. 3. 3. According to their pH optimum, blood plasma and seminal plasma α-glucosidases were typically neutral and acid enzymes and significant differences were recorded in their physico-chemical properties, establishing the tissue specificity of the enzyme. 4. 4. Notwithstanding the tissue under study, the nature of the α-glucosidase activity was similar in both strains of pure-bred rams.

An improved theory is presented for predicting the solute gradients that can be generated with vessels of variable or constant cross-sectional area together with flexible connecting tubing. Formulae are given for two possible arrangements using four such vessels and, where there is the restriction of identical starting volumes, equations are given for n compartments. The general principles behind the use of these equations for predicting pH gradients are considered, and the special case of two vessels is shown to be capable of generating eight different pH profiles as a function of volume delivered. Experiments are reported which support the usefulness of the theoretical treatment.

Two forms of Ca ++ -activated neutral protease (CANP) were found in the soluble fraction of bovine platelets and were partially purified, using (NH 4 ) 2 SO 4 fractionation and DEAE-Sepharose CL-6B, Sephadex G-150 chromatography. The activity of CANP was separated into two distinct forms (CANP-I & II) by DEAE-Sepharose chromatography. Both CANPs possessed very similar pH profiles (optimum pH 7.0) and sensitivities to protease inhibitors. Both CANPs required Ca ++ strictly for the activity, whereas the concentration of Ca ++ for the maximal activity was significantly different. CANP-I was fully activated in the presence of physiological range of free Ca ++ (10 −5 M), while CANP-II required at least 10 −3 M free Ca ++ for its maximal activity.

SummaryBy resuspending casein micelles in whey and dialysate it is shown that the role of whey proteins in the ethanol (EtOH)-induced coagulation of skim-milk is minimal. Experiments involving the interchange of milk sera indicated that the position of the EtOH stability/pH profile along the pH axis was governed by the diffusible constituents of the milk serum phase. The identities of those serum components governing the shape and position of the EtOH stability/pH profile were investigated. The addition of Ca2+ caused a shift in the entire profile to higher pH. Reduction of the available Ca2+ by addition of EDTA (up to 5 ran) shifted the profile to lower pH. The addition of phosphate (up to 5 mM) or citrate (up to 1 mM) had no effect on the profile, though higher concentrations of citrate (up to 5 mMi) caused slight shifts to lower pH. When equimolar amounts of Ca and phosphate were added, the system showed a shift in profile approximately equivalent to that of the free Ca introduced. Increasing the ionic strength of a milk by the addition of NaCl did not shift the profile, but decreased the maximum EtOH stability of the high pH arm of the sigmoidal profile. The EtOH stability/pH profile retained the same sigmoidal shape in all cases.

A new technique for generating extended pH gradients (3-4 pH units) in Immobiline gels for isoelectric separations is described. A five-chamber gradient mixer has been built, based on the 'Varigrad'-type mixers of Peterson and Sober (Anal. Chem. 31, 1959, 857-862). Each chamber contains one of the following Immobilines, in this order: pK values 4.4, 4.6, 6.2, 7.0 and 8.5, titrated in the pH 4-8 interval with non-buffering Immobilines pK 9.3 (in the case of the two acidic Immobilines) and pK 3.6 (in the case of the three basic Immobilines). In this way it is possible to cast, in a highly reproducible way, an immobilized pH gradient in the pH range 4.0 to 7.5, which should be ideal for isoelectric separations in the first dimension of two-dimensional techniques. A computer program is also described which, given the molarities and pK values of the different Immobilines in the chambers of the Varigrad mixer, can generate the theoretical pH profile, together with the buffering capacity (beta) and ionic strength (I) courses.

The effect of diffusion on the pH and temperature profiles of immobilized enzymes has been quantitatively assessed using the theory of pore diffusion. The pH profiles of immobilized enzyme systems varied from those of soluble ones to the square root of the relative values of the soluble ones depending on the degree of pore diffusion limitation. Also the decrease in activation energy was predicted quantitatively with considerable accuracy in terms of effectiveness factor. The experimental pH profiles of a few immobilized enzymes were compared with those theoretically predicted.

Summary The effect of toxin concentration on the kinetics of haemolysis of rabbit erythrocytes has been studied at pH values differing by about 0·5 pH units over the range 5·5 to 8·5 for both crude and partially purified toxin at concentrations of 2·5, 5·0, 10, 20, 30, 40, 60 MHD per ml and at 80 and 120 MHD per ml for the partially purified toxin. Increased concentration of toxin caused a decrease in the prelytic lag time (·) of the sigmoid haemolysis curves, a decrease in the time to reach 50 per cent. haemolysis (t1/2), and an increase in the maximum rate of haemolysis (Rm). Plateaux in Rm-toxin concentration, ·-toxin concentration and t1/2-toxin concentration curves were found at all pH values: these were displaced to higher concentrations in plots drawn for the partially purified toxin. The effect of pH on haemolysis is very marked, and pH profiles for the maximum rate of haemolysis were constructed: for the crude toxin, the shape of the profile changed as the toxin concentration was increased, while for the partially purified toxin the shape was independent of toxin concentration. The pattern of the profiles indicates that the crude toxin contains a small concentration of impurity that is strongly lytic at pH 5·5 and less markedly so at 8·5; the impurity has no significant effect at intermediate pH values. The reaction is first order with respect to toxin over a very small range of concentrations only, and the maximum rate of haemolysis is independent of toxin concentration at concentrations greater than 120 MHD per ml. The reaction is first order with respect to erythrocytes at all pH values, and over all measurable concentrations.

The pH dependence of electron transport in isolated lettuce chloroplasts can be simulated by a model involving four ionizable groups: two facing the inside and two facing the outside of the thylakoids. The pH dependence can be resolved into separate pH profiles for the outside and the inside of the thylakoids, with optima at 7.1 and 5.6, respectively. A ΔpH of 3 units across the thylakoid membrane allows only partial overlapping of the two profiles and relatively low rates of electron transport. Uncouplers stimulate electron transport by reducing the ΔApH, thereby improving the overlapping of the two pH profiles.

The methods used for the assay of lipoxidase have been compared. The pH profile of the enzyme, as measured by two of the methods, was found to be considerably altered by changes in the ionic strength of the reaction medium.

Separation of proteins or amino acids by isoelectric focusing in multicompartment devices has been proposed for large-scale purifications of biological mixtures. In the perspective of industrial applications, the present authors built a multicompartment apparatus and studied the pH profiles stabilized by simple non-amphoteric buffers (acetic acid and sodium acetate). Mixtures of two amino acids were separated to test this device. A theoretical model comprising one dimensionless separation parameter is proposed to characterize these separations. This model allows one to calculate the purity of the recovered amino acids, the yield of a separation at steady-state or the time necessary to obtain a given concentration of an amino acid in one of the compartments of the isoelectric focusing cell. The separation parameter contains the physical parameters which intervene in the electric migration and in the diffusion. Values of this separation parameter have been experimentally determined for three amino acids under various experimental conditions. The results confirm the usefulness of this model in designing a multicompartment isoelectric focusing apparatus.

The defoaming activity of three tablet antacids (hydrotalcite, hydrotalcite/dimethicone and aluminium hydroxide/dimethicone) powdered to 60 mesh was measured in vitro using a new static/dynamic technique. Their antacid actions and that of aluminium hydroxide gel B.P.C. were also measured using a modified Fuch's test. Combination of dimethicone with hydrotalcite conferred good defoaming activity with little effect on pH profile whilst combination of aluminium hydroxide with dimethicone markedly altered both. Additionally, the defoaming actions of the three commercial antacids were assessed in vivo. Radiographs were taken after administration of antacid, a foaming mixture and a normal barium meal. The radiographs were then ranked blind by 5 radiologists. The rankings assigned the significantly greatest (Mann-Whitney U-test) defoaming effect to the hydrotalcite/dimethicone combination, there being no difference between the other two preparations. The in vitro results were thus confirmed.