Range Of Buffer Concentrations Research Articles

Kinetics of Crude Peroxidase from the Rind of Watermelon Fruit

Aims: To study the kinetics of crude peroxidase from the rind of watermelon fruit in various assay conditions.
 Study Design: In vitro enzyme assay.
 Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between October 2015 and January 2016.
 Methodology: The activity of the crude peroxidase extracted from the rind of watermelon was determined by measuring the rate of oxidation of KI at 25oC in a 3.0 ml reaction mixture which contained 2.3 ml of 25 mM - 400 mM sodium acetate buffer (pH 3.5-6.0), 0.2 ml of 2 mM KI, 0.1ml of the crude peroxidase, and 0.2 ml of varying concentrations of chlorpromazine (0.01 mM - 0.1 mM). In all cases, 0.2 ml of 0.01 mM – 1 mM H2O2 was added last to initiate the reaction. Only one parameter was varied per assay. Assays were done in five replicates. The initial velocity of the crude peroxidase for KI oxidation was determined using the absorbance at 353 nm.
 Results: The concentration of H2O2 that generated an optimal activity for the crude peroxidase extracted was 0.2 mM, while a pH of 5.5 was optimal for the crude enzyme. The activity of the crude enzyme increased proportionately within a buffer concentration range of 25 mM and 400 mM. Chlorpromazine (0.01 mM - 0.1 mM) proportionately increased the enzyme activity, while promethazine within a range of 0.01 mM and 0.06 mM proportionally increased the enzyme activity. Further increase in promethazine concentration beyond 0.6 mM resulted in a decreased activity of the enzyme.
 Conclusion: This study suggests that the Rind of watermelon is an alternative source of peroxidase. The activity of this peroxidase can be enhanced by high buffer concentrations in the presence of some redox mediators like promethazine and chlorpromazine at a pH of 5.5.

Dynamics of Large DNA Loops

Dynamics of Large DNA Loops Long range interactions between genetically distant regions of DNA (>10 kbp) are not governed by the elastic energies of bending and twisting of DNA, but rather by entropic forces. Furthermore, such large scale interactions are mediated strongly by the excluded volume interactions of DNA and proteins of the cellular environment. It is not clear how these interactions locally couple to variables such as temperature, salt strength, pH, etc. in nanoconfined volumes where the monomer density of DNA is comparable to that in the cell nucleus. Using a nanofluidic device, we manipulate fluid flow to drive DNA into large loops, on the order of kilobasepairs, in a range of buffer concentrations and protein backgrounds. By analyzing the dynamics of the loop formation, steady state fluctuations, and deformation, we quantify an energy landscape. We can use this energy landscape to characterize activation barriers for proteins to form DNA loops. Once these activation barriers are overcome, we can test for enhancements in the loop elongation rate due to proteins. In addition, we can test for arrested states caused by proteins binding to specific sequences.

Bionanoparticles as candidate reference materials for mobility analysis of nanoparticles.

We propose bionanoparticles as a candidate reference material for determining the mobility of nanoparticles over the range of 6 × 10(-8)-5 × 10(-6) m(2)V(-1)s(-1). Using an electrospray differential mobility analyzer (ES-DMA), we measured the empirical distribution of several bionanoparticles. All of them show monomodal distributions that are more than two times narrower than the currently used calibration particles for mobility larger than 6 × 10(-8) m(2)V(-1)s(-1) (diameters less than 60 nm). We also present a numerical method to calculate corrected distributions of bionanoparticles by separating the contribution of the diffusive transfer function. The corrected distribution is about 20% narrower than the empirical distributions. Even with the correction, the reduced width of the mobility distribution is about a factor of 2 larger than the diffusive transfer function. The additional broadening could result from the nonuniform conformation of bionanoparticles and from the presence of volatile impurities or solvent adducts. The mobilities of these investigated bionanoparticle are stable over a range of buffer concentration and molarity, with no evidence of temporal degradation over several weeks.

The adsorption of Naproxen enantiomers on the chiral stationary phase Whelk-O1 under reversed-phase conditions: The effect of buffer composition

The adsorption of the Naproxen enantiomers on the chiral stationary phase ( S,S)-Whelk-O1 from methanol–water 80:20 (v/v) solutions modified with the addition of acetic acid or an acetic acid–sodium acetate buffer was studied using elution chromatography. Adsorption was found to be best accounted for by a two-site model assuming different retention mechanisms for the two enantiomers. Under experimental conditions causing a considerable degree of solute dissociation, strong distortion of overloaded band profiles is observed. This phenomenon is explained by the superimposition of the adsorption and the dissociation equilibria. The effect of the buffer composition on the retention is discussed and the results compared with previous ones obtained with the same system. The proposed model explains all the principal features of the adsorption of Naproxen on Whelk-O1 that were found earlier. Moreover this model applies well in a wider range of buffer concentrations, encompassing both the eluents in which solute dissociation is suppressed and those in which dissociation is significant.

Adsorption Behavior of Penicillin G Sodium on Hydrophilic Gel Toyopearl HW-40F

The adsorption of penicillin G sodium (PGS) on a hydrophilic gel, Toyopearl HW-40F, was investigated by the frontal analysis method. Effects of media, buffer concentration, and temperature on the adsorption equilibrium were examined in the buffer concentration range of (0.005 to 0.05) mol·L−1 and temperature range of (278.15 to 308.15) K. At the same pH value, the ionic strength of the mobile phase greatly influences the adsorption of PGS. The adsorption isotherms of PGS are comparable at different temperatures. The equilibrium data were modeled by the Freundlich equation with AARD in the range of (0.86 to 7.15) %.

Substrate specificity and some physicochemical properties of autolytic enzymes of the bacterium Lysobacter sp. XL 1

The substrate specificity of autolytic enzymes of the bacterium Lysobacter sp. XL 1 has been established. The periplasmic enzyme A8, the cytosolic enzyme A1, and the enzyme A10 solubilized from the cell walls and membranes with Triton X-100 exhibit glucosaminidase activity; the cytosolic enzyme A4 and the enzyme A9 solubilized from the cell walls and membranes with LiCl exhibit the muramidase activity. The cytosolic enzymes A3 and A6 have N-acetylmuramoyl-L-alanine amidase activity, and the enzyme A5 exhibits the diaminopimelinoyl-alanine endopeptidase activity. Some physicochemical properties of the most active autolytic cytosolic enzymes of Lysobacter sp. XL 1 (endopeptidases A5 and A7 and N-acetylmuramoyl-L-alanine amidase A6) were studied. The enzymes exhibit maximal activity over a wide range of buffer concentrations in weakly alkaline medium and moderate temperatures. The investigated enzymes are comparatively thermolabile proteins.

Direct electrochemical detection of sodium azide in physiological saline buffers using highly boron-doped diamond electrodes

Direct detection of sodium azide was examined by using highly boron-doped diamond (BDD) electrodes in some physiological saline buffer solutions. Three important saline buffers, ADA buffer ( N-(2-acetamido) iminodiacetic acid), sodium EDTA and imidazole, were investigated. Hydrogen-terminated BDD was fixed for the investigation since it showed much better sensitivity for azide oxidation in comparison with the oxygen-terminated one. Wide range of buffer concentration was examined. A well-separated peak of sodium azide was observed in its mixture with the buffers, resulting in selective sodium azide detection. The current responses were exactly total addition of sodium azide and buffer current responses. The present method was evidenced to be available for direct detection of sodium azide in two samples of commercial diluents for cell counting. A standard addition method was applied. Sodium azide concentrations of 502 and 1355 μg mL −1 were detected in both samples with the R.S.D.s of 0.07 and 2.37%, respectively. Validation was achieved by the comparison with the sample concentrations given by the manufacturer. Simple, selective, good sensitivity and excellent reproducible detection make the diamond electrodes very attractive for numerous electroanalytical applications.

Influence of buffer and l-phenylalanine concentration in the Rhizomucor miehei lipase catalysed synthesis of l-phenylalanyl-d-glucose ester investigated through response surface methodology

Reaction conditions for Rhizomucor miehei lipase (RML) catalysed synthesis of l-phenylalanyl-d-glucose using unprotected l-phenylalanine and d-glucose were optimized using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed involving 32 experiments of five variables (l-phenylalanine concentration in mmol, amount of RML in mg, pH, incubation period in h and buffer concentration in mM) at five levels. A second-order polynomial equation was developed in terms of linear, quadratic and cross product terms to study the effects of variables on esterification yields. A R 2 value of 0.7 was obtained for this complex reaction. From the surface and contour plots it was found that higher yields were observed for a very narrow pH range of 4.5–6.5 at l-phenylalanine concentrations above 3 mmol. The extent of esterification decreased with increase in RML concentration for incubation periods below 60 h. However, longer incubation periods above 60 h, enhanced esterification at all RML concentrations. Lower conversions below 0.5 mmol required less than 3.5 mmol l-phenylalanine concentration and a broad range of buffer concentration from 0.1 mM (0.1 ml, 0.1 M buffer of pH 6.0) to 0.5 mM (0.5 ml, 0.1 M buffer of pH 6.0). However, higher conversions from 0.6 to 0.8 mmol required a buffer concentration above 0.25 mM (0.25 ml, 0.1 M buffer of pH 6.0) at l-phenylalanine concentrations above 3.5 mmol. An optimum predicted yield of 1.01 mmol for l-phenylalanyl-d-glucose at 3 mmol l-phenylalanine, 100 mg of RML, 24 h incubation period, 0.5 mM (0.5 ml of 0.1 M buffer), pH 4.8 acetate buffer was found to agree with 0.97 mmol obtained under these experimental conditions. Validation experiments carried out under random conditions also exhibited good correspondence between predicted and experimental yields.

Adsorption of alpha-chymotrypsin onto mica in laminar flow conditions. Adsorption kinetic constant as a function of tris buffer concentration at pH 8.6.

We examined the adsorption kinetics of alpha-chymotrypsin (pH 8.6, 10(-2) to 0.5 M Tris buffer) on muscovite mica in conditions of laminar flow through a slit. The range of buffer concentrations is between two limits: (i) no adsorption in 1 M Tris and (ii) no desorption in 10(-3) M Tris. Studying the dependence of adsorption kinetics on the wall shear rate leads to the determination of the interfacial adsorption kinetic constant ka and the diffusion coefficient. The obtained value for the diffusion coefficient is close to the one expected from the molecular size of alpha-chymotrypsin. The interfacial adsorption kinetic constant of alpha-chymotrypsin decreases when ionic strength increases, while the initial desorption constant (over a part of all the adsorbed population) shows the contrary. Although alpha-chymotrypsin is almost at its isoelectric point, the effect of ionic strength on the adsorption kinetics suggests the importance of electrostatic interactions between the protein and mica. We observed an increase in the adsorption rate, at a surface coverage near 0.14 microg cm(-2), for adsorption in 10(-2) M Tris and the low wall shear rates (<300 s(-1)). This change in the adsorption rate suggests a structural transition, that we assume again to be due to electrostatic interactions, but between proteins. The large dipole moment of the protein may induce this transition, illustrated here by the ferroelectric/antiferroelectric pattern. The variation of the zeta potential with interfacial concentration seems to be in agreement with such a model.

Effect of buffer concentration on gradient chromatofocusing performance separating proteins on a high-performance DEAE column

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of β-lactoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chromatofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, β-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of β-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 m M (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 m M (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.

The pH of buffers based on 2-amino-2-hydroxymethyl-1,3-propanediol (‘tris’) in synthetic sea water

E.m.f. measurements were made over the temperature range 273.15–318.15 K, and at five salinities from 20 to 40, using the cell Pt( s) H 2( g, p°) BH + and B in synthetic sea water AgCl( s) Ag( s) Pt( s) where p°=101.325 kPa, and BH + and B are the conjugate acid–base pair of 2-amino-2hydroxymethyl-1,3-propanediol (‘tris’) – each present at a concentration of 0.04 mol kg -1. The results were used to assign pH values based on the ‘total’ hydrogen ion scale to such buffer solutions based on this acid–base pair. Additional measurements were made over a range of buffer concentrations at a salinity of 35; these were used to calculate the stoichiometric (ionic medium) dissociation constant for ‘tris–H +’ in synthetic sea water.

The effects of acetate buffer concentration on lysozyme solubility

The micro-solubility column technique was employed to systematically investigate the effects of buffer concentration on tetragonal lysozyme solubility. While keeping the NaCl concentrations constant at 2%, 3%, 4%, 5% and 7%, and the pH at 4.0, we have studied the solubility of tetragonal lysozyme over an acetate buffer concentration range of 0.01M to 0.5M as a function of temperature. The lysozyme solubility decreased with increasing acetate concentration from 0.01M to 0.1M. This decrease may simply be due to the net increase in solvent ionic strength. Increasing the acetate concentration beyond 0.1M resulted in an increase in the lysozyme solubility, which reached a peak at ∼ 0.3M acetate concentration. This increase was believed to be due to the increased binding of acetate to the anionic binding sites of lysozyme, preventing their occupation by chloride. In keeping with the previously observed reversal of the Hoffmeister series for effectiveness of anions in crystallizing lysozyme, acetate would be a less effective precipitant than chloride. Further increasing the acetate concentration beyond 0.3M resulted in a subsequent gradual decrease in the lysozyme solubility at all NaCl concentrations.

Effects of buffer concentration on the electrophoretic behaviors of small peptides in capillary zone electrophoresis

AbstractIt was found that unlike the electroosmotic flow, the electrophoretic mobilities of small peptides in capillary zone electrophoresis (CZE) were invariant over the buffer concentration range studied. Migration of these peptides in CZE is a electroosmotic‐governed process. The quantitative linear relationships between the logarithm of migration times (log tm) and the reciprocal of column temperature were investigated under different buffer concentrations to study the activation energies of diffusion (AED) as the function of the buffer concentration. The preexponential factors were quantitatively correlated with the structural parameters of the peptides studied. © 1995 John Wiley &amp; Sons, Inc.

Interferences, limitations and an improvement in the extraction and assessment of cellulase activity in soil

A practical modification of areducing-sugar method to estimate soil (carboxymethyl) cellulase activity is described and used to compare three non-sterile soils. The original method involves the formation of a ferric-ferrocyanide complex in soil extracts prepared and exposed to substrate in 2 M acetate buffer. Because high acetate concentrations compromise this reaction, such extracts require considerable (e.g. 20–30-fold) dilution prior to activity determinations. For soils with low organic matter, such as one used in this study, undetectable activity often results. We used 67 mM acetate buffer and were able to detect and compare cellulase activities in three diverse soils using undiluted extracts. The values obtained were more reproducible, but in a different range from those of the original method. Our modification yielded activities that appeared to be unchanged over the extraction buffer concentration range of 11–140 mM acetate. The use of undiluted soil extracts prepared in less concentrated buffer simplifies and extends the practicality of the assay by: (a) reducing the importance of using matrix-matched standard curves; (b) requiring less sample manipulation and glassware, and (c) preventing the dilution to extinction of enzyme activity. It also improves the usefulness of the method as an indicator of ecological effects (biomass turnover) due to the introduction of nonindigenous microorganisms and chemicals of environmental concern. These factors make the method more competitive with other reducing-sugar assays used to measure cellulase activity in soil.

On the mathematical model of capillary electrophoresis

The influence of the electroosmotic flow profile on the efficiency of capillary electrophoresis for a wide range of buffer concentrations and capillary diameters is studied. Thermal effects in the capillary, the finite size of solute molecules and their interactions with the capillary wall are also taken into consideration. The dependence of capillary electrophoresis efficiency and resolution on the zeta-potential values is investigated.

Investigation of the electroosmotic flow effect on the efficiency of capillary electrophoresis.

The influence of the electroosmotic flow profile on the efficiency and resolution of capillary electrophoresis is studied. The mathematical model is formulated and the set of equations is solved numerically. The results of the analysis are applicable to a wide range of buffer concentrations and capillary diameters. The temperature dependence of electrophoretic mobility, viscosity and thermal conductivity and the dependence of electrical conductivity on temperature and ion concentration in the double layer are taken into account. It is shown that there exists a region of buffer concentrations and capillary diameters where the influence of the electroosmotic flow profile on the efficiency and resolution is much greater than that of the temperature dependence of the electrophoretic mobility. The results are especially essential for small buffer concentrations or capillary diameters comparable with the double electrical layer thickness.

Deposition of polystyrene particles in a parallel plate flow cell. 1. the influence of collector surface properties on the experimental deposition rate

Deposition of polystyrene particles on different materials with various surface free energies has been studied as a function of the concentration of a potassium phosphate buffer. The results are correlated with theoretical calculations based on the DLVO theory and taking into account prevailing hydrodynamic conditions. Generally it was found that deposition rates increase gradually as a function of the buffer concentration, contrary to the theory, predicting a steep increase at a critical ionic strength. This discrepancy is ascribed to surface heterogeneity and polymer hairs protruding from the particle surface. Furthermore, the collector surface free energy greatly influenced the difference between the theoretically calculated and experimentally measured buffer concentration beyond which deposition starts to increase steeply. For a low surface free energy of the collector a large difference is observed, whereas for a high surface free energy of the collector small differences were observed between theory and experimental observations within the range of buffer concentrations applied.

Separation of membrane proteins of H. halobium by gel permeation high performance liquid chromatography.

High performance liquid chromatography (HPLC) separation of the SDS-solubilized membrane proteins of Halobacterium halobium S9 was investigated with a gel permeation column, TSK G3000 or 4000 SW. When sodium phosphate buffer (pH 7.0) containing 0.1% SDS was used for elution, both the elution volume and the elution profile of protein peaks were highly dependent on the buffer concentration. On increasing the buffer concentration, the elution volume for each increased and the resolution was also improved. Protein was recovered quantitatively from the column in the buffer concentration range between 0.01 to 0.2 M. In contrast to the phosphate buffer system, the elution profile of the membrane protein was almost independent of the salt concentration when Tris-HCl (pH 7.0) buffer containing 0.1% SDS and various amounts of NaCl was used for elution. The best separation conditions finally determined for the membrane proteins of H. halobium using SW columns were as follows : 0.2 M Tris-HCl (pH 7.0) containing 0.1 M NaCl and 0.1% SDS as an elution buffer system, at a flow rate of 0.6 ml per min. Under the above elution conditions, nine major protein peaks could be determined in the SDS-solubilized plasma membrane preparation of H. halobium S9 within 40 min when the absorbance was recorded at 280 nm. The resolution obtained is comparable to that of SDS-PAGE.

Association of phycoerythrin and phycocyanin: in vitro formation of a functional energy transferring phycobilisome complex of Porphyridium sordidum.

Functional in vitro association and dissociation of a phycobiliprotein complex, isolated from phycobilisomes of the red alga Porphyridium sordidum, were studied. The complex contained large bangiophyceaen phycoerythrin and cyanophytan phycocyanin in an equimolar ration and had absorption maxima at 625, 567, and 550 nm and a shoulder at 495 nm. Emission at 655 nm (with excitation at 545 nm) from phycocyanin indicated functional coupling. The complex was stable over a wide buffer concentration range, and, notably, it was maximally stable in low phosphate, less than 0.01 M, unlike the phycobilisomes, which dissociate at this concentration. Its molecular weight was estimated to be ca. 510000, and by electron microscopy it was seen to consist of two units of similar size. The complex in 0.1 M phosphate was separated on a sucrose gradient into a homogeneous phycoerythrin band and a spectrally heterogeneous phycocyanin band. In vitro association of phycoerythrin and phycocyanin resulted in a complex with the same absorbance, emission, sedimentation and molar pigment ratio as those of the native complex. The spectrally heterogeneous phycocyanin fractions from the dissociation gradient varied in the degree of association with phycoerythrin. Phycocyanin fractions absorbing from 622 to 633 nm exhibited high associability (greater than 70%), whereas those with maxima at 617-620 nm had low associability (less than 30%). The presence of a 30000 molecular weight polypeptide accompanied high associability, where it was ca. 2-fold more prominent. It is suggested that this polypeptide is involved in complex formation and could serve either in the stabilization of the conformational state of cyanophytan phycocyanin or as a direct linker between phycobiliproteins.

Systems approach to vaginal delivery of drugs IV: methodology for determination of membrane surface pH.

A physical model including a diffusional layer in series with the membrane was developed for studying the possible differences between the pH at the membrane surface and that in the bulk solution. Both the membrane-secreted substances (acids and bases) and buffer constituents in the bulk solutions are assumed to contribute to the surface pH. Equations derived for this situation, together with experimental determinations of the acidic dissociation constant of the secreted material, the total secretion flux, the flux of total secreted acidic species, and the diffusion layer thickness, allow estimates to be made of the pH at the membrane surface. With the rabbit vagina, the membrane surface pH was close to that of the bulk solution in most cases. These results were supported by the fact that the absorption of 1-alkanoic acids in pH 2.2 phosphate buffers was relatively constant over the buffer concentration range of 0.003-0.1 M phosphate.