Potassium Phosphate Buffer Solution Research Articles

Protein Denaturation during Freezing and Thawing in Phosphate Buffer Systems: Monomeric and Tetrameric β-Galactosidase

During freezing in sodium and potassium phosphate (NaP and KP) buffer solutions, changes in pH may impact the stability of proteins. Since the degradation pathways for the model proteins, monomeric and tetrameric β-galactosidase (β-gal), chosen for this study are governed by conformational changes (i.e., physical instability) as opposed to chemical transformations, we explored how the stresses of freezing and thawing alter the protein's native structure and if preservation of the native conformation during freeze–thawing is a requisite for optimal recovery of activity. During freezing in NaP buffer, a significant pH decrease from 7.0 to as low as 3.8 was observed due to the selective precipitation of the disodium phosphate; however, the pH during freezing in KP buffer only increased by at most 0.3 pH units. pH-induced inactivation was evident as seen by the lower recovery of activity when freeze–thawing in NaP buffer as compared to KP buffer for both sources of β-gal. In addition, we investigated the effects of cooling rate and warming rate on the recovery of activity for monomeric and tetrameric β-gal. Optimal recovery of activity for the NaP samples was obtained when the processing protocol involved a fast cool/fast warm combination, which minimizes exposure to acidic conditions and concentrated solutes. Alterations in the native secondary structure of monomeric β-gal as measured by infrared spectroscopy were more significant when freezing and thawing in NaP buffer as opposed to KP buffer. Conformational and activity analyses indicate that pH changes during freezing in NaP buffer contribute to denaturation of β-gal. These results suggest that proteins formulated in NaP buffer should be frozen and thawed rapidly to minimize exposure to low pH and high buffer salts.

Infrared Absorption and Ultraviolet-Circular Dichroism Spectral Studies of Thermally Induced Unfolding of Apomyoglobin

The static infrared absorption (IR) and circular dichroism (CD) spectra of the temperature-dependent conformational states of apomyoglobin in potassium phosphate and sodium acetate buffer solution at pH 5.3 in D2O are reported. The circular dichroism ellipticity at 222 nm reveals a loss of helical structure for both heat- and cold-denatured states. Cold denaturation leads to a molten globule state with an associated partial loss of helical structure identified at 1646 cm−1. Heat denaturation also exhibits an absorption loss at 1646 cm−1 but leads to the appearance of aggregation at 1680 and 1619 cm−1. There is semi-quantitative agreement of the ultraviolet circular dichroism and infrared estimates of protein helicity. The denaturation spectra give evidence that there are specific associations between acetate ions and the protein, but not between phosphate ions and the protein.

Stability-indicating high-performance liquid chromatographic assay of buspirone HCl.

The United States Pharmacopoeia high-performance liquid chromatographic (HPLC) assay method of buspirone is not able to discriminate buspirone from its degradation products. The purpose of this work is to develop a sensitive, selective, and validated stability-indicating HPLC assay for the analysis of a buspirone hydrochloride in a bulk drug. Buspirone HCI and its potential impurities and degradation products are analyzed on an Ultrasphere C18 column heated to 40 degrees C using a gradient program that contains monobasic potassium phosphate buffer solution (pH 6.9) and acetonitrile-methanol mixture (13:17) of 35% for 5 minutes, then increased to 54% in 5.5 minutes. The samples are monitored using a photo-diode array detector and integrated at 244 and 210 nm. The stress testing of buspirone HCI shows that buspirone acid hydrochloride is the major degradation product. The developed method shows a separation of buspirone degradation product and its potential impurities in one run. The stability of buspirone HCI is studied under accelerated conditions in order to provide a rapid indication of differences that might result from a change in the manufacturing process or source of the sample. The forced degradation conditions include the effect of heat, moisture, light, acid-base hydrolysis, sonication, and oxidation. The compatibility of buspirone HCI with some pharmaceutical excipients is studied under stress conditions. The linear range of buspirone HCI is between 5 and 200 ng/microL with a limit of quantitation of 2.5 ng/microL. The intraassay percentage deviation is not more than 0.38%, and the day-to-day variation was not more than 0.80%. The selectivity, repeatability, linearity, range, accuracy, sample solution stability, ruggedness, and robustness show acceptable values.

Flow-injection immunoassay using OPA derivatization

A flow-injection immunoassay (FIIA) using ortho-phthalaldehyde (OPA) derivatization was developed for on-line monitoring of protein products in biotechnological processes. Recombinant tissue-type plasminogen activator (rt-PA) and its antibodies were used in these studies as a model system. The derivatization of rt-PA with OPA led to a lower detection limit (higher fluorescence intensity) and an increase of sensitivity (higher slope of calibration curve) for rt-PA compared with the method without OPA derivatization. The derivatization of rt-PA with OPA was also systematically characterized: influence of pH, derivatization time, OPA concentration and sample volume on fluorescence intensity. Interferences caused by proteins were investigated with different washing buffer solutions. A 0.1 M potassium phosphate buffer (PPB) solution containing 1 g l −1 polyethylene glycol (PEG) was found to be optimal for minimizing the interference with BSA in the measurement. On-line monitoring for rt-PA is carried out on a simulated bioprocess in the range 0–20 μg ml −1. The data obtained showed good agreement with the conventional off-line enzyme linked immunosorbent assay (ELISA) data. The FIIA using o-phthalaldehyde (OPA) derivatization is a valuable tool for on-line monitoring of low concentrated protein products in biotechnical production processes.

High-performance liquid chromatography of selected organic peroxides with oxidative amperometric detection.

Reversed-phase high-performance liquid chromatography with oxidative amperometric detection was optimized for the determination of several organic peroxides in drinking water under ideal conditions. The determinations were performed under isocratic conditions using acetonitrile and methanol as the organic modifiers with 0.05 M potassium phosphate buffer solution. The oxidative amperometric response of the organic peroxides was dependent on the concentration of organic modifier and the electrode potential. The optimum electrode potential (EOxAPP), for the simultaneous determination of the organic peroxides was approximately +1.150 +/- 0.05 V versus the Ag/AgCl reference electrode. The maximum analytical signal for butan-2-one peroxide and tert-butyl hydroperoxide, when using acetonitrile, was obtained with 20% v/v organic modifier. For cumene hydroperoxide, the maximum analytical signal was achieved with approximately 35% v/v acetonitrile. The retention time of cumene hydroperoxide, on an octyldecylsilane column (250 x 4 mm id), decreased sharply from > 100 to < 10 min when the organic modifier concentration was varied from 5 to 50% v/v. The retention time of butan-2-one and tert-butyl hydroperoxide, under the same conditions, varied by < 10 min. The calibration curves for the aliphatic peroxides and aromatic peroxide were linear from 2 to 200 ng and from 0.2 to 200 ng injected, respectively.

Injectable cartilage.

Slowly polymerizing calcium alginate gels were investigated as a means of delivering large numbers of isolated chondrocytes by means of injection to determine if these gels would promote engraftment and could provide three-dimensional templates for new cartilage growth. Chondrocytes isolated from the articular surface of calf forelimbs were added to a 1% sodium alginate dissolved in a 0.1 M potassium phosphate buffer solution (pH 7.4) to generate a final cellular density of 10 x 10(6)/ml (representing approximately 10 percent of the cellular density of human juvenile articular cartilage). The calcium alginate-chondrocyte mixture was injected through a 22-gauge needle in 100-microliters aliquots under the panniculus carnosus on the dorsum of nude mice and incubated for 6 (n = 4), 8 (n = 11), and 12 (n = 12) weeks in vivo. Time-zero specimens (n = 10) consisting of 100-microliters aliquots of the calcium alginate-chondrocyte mixture were used to calculate initial weight. At harvest, all calcium alginate-chondrocyte specimens exhibited a pearly opalescence and were firm to palpation as early as 6 weeks after injection. By 12 weeks of in vivo incubation, the specimens weighed 0.15 +/- 0.04 gm, significantly more than the initial weight of 0.11 +/- 0.01 gm (p < 0.05). Specimens stained with hematoxylin and eosin demonstrated lacunae within a basophilic ground-glass substance. Control specimens of calcium alginate without chondrocytes (n = 4) had a doughy consistency 12 weeks after injection and had no histologic evidence of cartilage formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of aromatic l-amino acid decarboxylase from liver of skipjack tuna (Katsuwonus pelamis, Teleostei: Scombridae)

Aromatic l-amino acid decarboxylase (AADC; EC 4.1.1.28) was extracted from skipjack (Katsuwonus pelamis) liver with potassium phosphate buffer solution. This enzyme was purified by ammonium sulfate fractionation, sepharose CL-6B, QAE-Toyopearl 550C, DEAE-Toyopearl 650M, Bio-gel HT, and Toyopearl HW-55F chromatography. The molecular weight of the enzyme was estimated to be about 110,000 by gel filtration on Sepharose CL-6B. The enzyme was inhibited by Co2+, Cu2+ and Zn2+ ions, but activated by K+, Li+, Na+, Ba2+, and Mn2+ ions.

Kinetics of Electrochemical Reduction of Carbon Dioxide on a Gold Electrode in Phosphate Buffer Solutions

Abstract The kinetics of the CO formation by electrochemical reduction of carbon dioxide on a gold electrode investigated in a series of potassium phosphate buffer solutions. CO is the principal product under the above condition. Small amounts of hydrocarbons, such as CH4, C2H4, and C2H6, are detected in the solutions of pH 5.2, 6.2, and 6.8. The Tafel slopes of the partial current density for the CO formation, ICO, at different pH are all ca. −120 mV/decade. The reaction mechanism has been proposed on the basis of the relationships between ICO and the electrode potential and between ICO and the reaction order with respect to CO2, which is ca. 1.

Dual enzyme amperometric biosensor for putrescine with interference suppression

AbstractA putrescine biosensor has been developed. Putrescine oxidase, peroxidase, and bovine serum albumin were cross‐linked directly on the surface of a graphite electrode by glutaraldehyde. The sensor was used to determine putrescine at 0 mV (vs. Ag/AgCl reference electrode) in a potassium phosphate buffer (pH 7.4) solution containing 4 mM dopamine (3‐hydroxytyramine). The response of putrescine was linear up to 600 μM with a standard deviation of 2.8%. The detection limit was 5 μM. The response time was around 30 s. The lifetime was over 30 days when the electrode was used every day. The interferences of cadaverine and spermidine could be eliminated by added dopamine which, apparently, inhibits the enzymatic conversion of these substrates more strongly than that of putrescine.

FTIR spectroscopic study of the dynamics of conformational substates in hydrated carbonyl-myoglobin films via temperature dependence of the CO stretching band parameters

Two hydrated carbonyl myoglobin (MbCO) films, one containing (0.30 g water)/(g MbCO) from MbCO solution in water at pH 5.5 and the other (0.32 g water)/(gMbCO) from 0.1 M potassium phosphate buffer solution at pH 6.8, were studied by FTIR spectroscopy from 293 K to 78 K at selected temperatures on cooling and reheating. Above approximately 180 K the general trend in temperature dependence of half-bandwidths, peak maxima, and band area ratios of the A1 and A3 conformer bands is similar to those reported by Ansari et al. (1987. Biophys. J. 26:337) for MbCO in 75% glycerol/water solution, but abrupt changes in slopes at approximately 180-200 K and freezing-in of conformer populations, which could be taken as indicator for glass transition of the solvent or the protein, are absent for the hydrated MbCO films. This is interpreted in terms of an exceptionally broad distribution of relaxation times, and is in accord with conclusions from recent calorimetric annealing studies of hydrated protein powders (Sartor et al. 1994. Biophys. J. 66:249). Exchange between the three A conformers does not stop at approximately 180-200 K but occurs over the whole temperature region studied. These results are then discussed with respect to MbCO's behavior in the glass-->liquid transition region of glass-forming solvents, and it is concluded that, in analogy to the behavior of low-molecular-weight compounds with a distribution of rapidly interconverting conformers, freezing-in of MbCO's A conformer populations by the solvent should not be mistaken for a glass transition of MbCO.

Isoflurane increases the anaerobic metabolites of halothane

The effect of isoflurane on the anaerobic metabolism of halothane to chlorodifluoroethene (CDE) and chlorotrifluoroethane (CTE) was studied with microsomes of guinea pig liver by gas chromatography. The reaction mixture used to measure the end products of anaerobic metabolism consisted of a microsomal suspension, 3 mM NADPH, halothane and isoflurane (except in control groups) in 0.1 M potassium phosphate buffer solution (pH 7.4). The Km values for CDE formation were 601.61 +/- 266.91, 254.22 +/- 86.58, 257.92 +/- 129.11, 268.55 +/- 125.66 and 319.22 +/- 86.76 microM (mean +/- SD, n = 5) at 0 mM (0%), 0.12 mM (0.26%), 0.29 mM (0.64%), 0.58 mM (1.30%) and 1.16 mM (2.59%) isoflurane, respectively. The Km values for CTE formation were 1204.74 +/- 551.64, 553.75 +/- 177.89, 521.14 +/- 249.77, 560.67 +/- 229.61 and 711.05 +/- 317.13 microM (n = 5) at 0 mM (0%), 0.12 mM (0.26%), 0.29 mM (0.64%), 0.58 mM (1.30%) and 1.16 mM (2.59%) isoflurane, respectively. In contrast, the Vmax values for CDE and CTE formation at these isoflurane concentrations were not significantly different than in the control groups. In this study the production of CDE and CTE was significantly (P < 0.05) increased by isoflurane, at concentrations up to 0.58 mM (1.30%).

Epidermal Growth Factor Interacts with Indole-3-Acetic Acid and Promotes Coleoptile Growth

We used coleoptile sections of Avena sativa, Sorghum bicolor, and Zea mays seedlings to examine interactions between epidermal growth factor (EGF) and indole-3-acetic acid (IAA) that may affect plant growth and development. Our 24-h bioassays employed three controls ranging in dilution from 10−4 to 10−8 g ml−1: (1) 50 mM potassium-phosphate buffer solution (pH=6.0), (2) bovine serum albumin, a nonspecific protein; and (3) IAA; plus two treatments: (1) mouse epidermal growth factor (EGF) ranging from 10−6 to 10−10gml−1, and (2) EGF + IAA. In all three species growth in IAA, EGF, and EGF + IAA treatments showed significant increases over controls; EGF+IAA showed significant increases in growth over IAA alone. As the concentrations of IAA decreased, the EGF and IAA interaction became more pronounced. At the highest IAA concentrations, EGF + IAA increased growth rates ca. 2% to 39%, whereas at lower IAA concentrations EGF + IAA promoted growth as much as 121%, thereby lowering the normal IAA physiological set point up to three or four orders of magnitude. Our data suggest that an interaction between EGF and IAA may allow plants to recognize and respond to animal biochemical messengers, resulting in changes in plant cell elongation that ultimately may alter plant growth patterns.

Selective separation and purification of two lipases from chromobacterium viscosum using AOT reversed micelles

Selective separation and purification of two lipases form Chromobacterium viscosum were carried out by liquid-liquid extraction using a reversed micellar system. Optimum parameters for extraction were determined using a 250 mM AOT micellar solution in isooctane. Complete separation of the two lipases was achieved at pH 6.0 with a 50mM potassium phosphate buffer solution containing 50 mM KCI. By adding 2.5% by volume of ethanol to the lipase-loaded micellar solution, 85% of the extracted lipase could be recovered in a new aqueous phase, 50 mM K(2)HPO(4) with 50 mM KCl, at pH 9.0. Lipase A was purified 2.6-fold with a recovery of 86%, and lipase B by 1.5-fold with a recovery of 76%.

Chemically modified electrodes for the determination of sulphydryl compounds

Under neutral conditions, the electrocatalytic oxidation of sulphydryl compounds at rotating graphite electrodes modified with tetracyano- p-quinodimethane, tetrathiafulvalene and 1,1′-dimethylferrocene proceeds at potentials higher than −0.1 V vs. SCE. The sensitivity of the electrodes depends on the nature of both the modifier and the sulphydryl compounds, and in 0.1 M potassium phosphate buffer solution (pH 7) it reaches 0.16 A 1 mol −1 cm −2. The electrode sensitivity is higher in a weakly alkaline medium. The sensitivity is only slightly dependent on the electrode rotation speed.

Biomechanical and biochemical changes in lumbar vertebrae of rapidly growing rats.

Although growth-related biochemical, morphological, and biomechanical properties of rat cortical bone have been investigated, similar properties of immature rat vertebral bone have not been well characterized. Information about these properties is necessary, however, for comparative analyses of rat vertebral bone adaptation. Thus a method was developed to characterize growth-related differences in immature rat vertebral bone. The centra of sixth lumbar vertebrae (L6) of 44-day-old and 54-day-old male Sprague-Dawley rats were compressed to 50% of their initial height at a 50%/s strain rate while immersed in a potassium phosphate buffer solution (pH 7.4, 37 degrees C). Structural and material properties for the 54-day-old group that were significantly greater than those for the 44-day-old group included load, stress, and energy at the proportional limit; initial maximum load and stress; and load and energy at 50% strain. The structural stiffness of L6, as well as its elastic modulus, was significantly greater in the older animals. The calcium concentrations and calcium-to-collagen ratios in 54-day-old vertebrae were significantly greater than in younger animals. These results indicated that the specimen preparation and testing protocol developed for rat vertebrae produced reliable biomechanical results, even with the relatively small size of rat vertebrae bodies, and that the quantity and quality of the matrix of immature rat vertebral bone changed significantly during this period of rapid growth. Our test protocol will be useful for investigating the responses of rat vertebral bone to exercise, disease, and spaceflight.

Dependence of autolysis of Bacillus subtilis cells on macromolecule synthesis under nutrient limitation

Autolysis of Bacillus subtilis 168 cells in buffer solution was induced by high concentrations of monovalent cations, which activate N- acetylmuramoyl- l-alanine amidase, a major autolysin in this bacterium. However, cells did not lyse in Spizizen salt medium containing high K + ion concentration. Therefore, autolysis of washed cells of B. subtilis in the log phase was studied in various nutrient solutions in the presence of 100 mM K +. Glucose inhibited autolysis in 100 mM KCl solution, but ammonium ions together with glucose were required to inhibit lysis in potassium phosphate buffer (KPB) solution. Penicillin G or vancomycin caused cell lysis in KPB + glucose + ammonium ion (KPB non-lytic solution), but not in KCl + glucose (KCl non-lytic solution). A tracer experiment showed that synthesis of both protein and peptidoglycan was active in KPB non-lytic solution but not in KCl non-lytic solution. Chloramphenicol-treated cells did not lyse in KPB non-lytic solution upon addition of penicillin G. Autolysis observed during active protein synthesis without peptidoglycan synthesis in KPB + glucose solution was suppressed by chloramphenicol. From these results, we concluded that autolysis occurred when protein synthesis was active but peptidoglycan synthesis was incomplete.

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,N′methylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca2+-alginate immobilized cells.

Chloroperoxidase Catalyzed Halogenation of Pyrimidine Bases

Abstract Treatment of uracil or cytosine with H2O2 and potassium halides in the presence of chloroperoxidase in potassium phosphate buffer solution at pH 3.0 resulted in halogenation of the substrates, whereas no reaction occurred on thymine by a similar treatment.

Isoproterenol-induced mortality and cardiac lipids of aspirin-pretreated rats with various fat intakes

The effect of altering cardiac concentrations of precursors and inhibitors of prostaglandin synthesis by varying fat intake was determined in rats injected with the cardiotoxic drug isoproterenol, following pretreatment with aspirin or potassium phosphate buffer solution. Prior to injection, four groups of rats were fed either a low-fat diet (3.7 energy percent coconut oil 3.7 energy percent safflower oil) or a high-fat diet (3.7 energy percent safflower oil-36.4 energy percent coconut oil mixture or 40.1 energy percent safflower oil.) Mortality as well as fatty acid composition of cardiac lipids changed in response to altered kinds and amounts of fats. Mortality and cardiac C20:4/C22:6 ratio were lowered by feeding 3.7 energy percent coconut oil, and increased by feeding 40.1 energy percent safflower oil. Aspirin reduced mortality in rats fed 40.1 energy percent safflower oil, but not in rats fed other diets. Results suggest that dietary manipulations which increase tissue content of polyunsaturated fatty acids of the n-6 type relative to those of the n-3 type may increase sensitivity to isoproterenol, and that effectiveness of aspirin in reducing isoproterenol-induced mortality depends upon the n-6/n-3 ratio of cardiac fatty acids.

Effects of Triethanolamine and Related Compounds on the Enzyme Activity of Glutamic Acid Dehydrogenase

An investigation was attempted to determine the effects of triethanolamine, monoethanolamine, ammonium sulfate, hydrazine hydrate and hydroxylamine hydrochloride on the enzyme activity of glutamic acid dehydrogenase which is a pyridine enzyme playing an important role in the oxidation and reduction of living bodies. Results obtained were as follows:1) Sodium glutamate and DPN were dissolved in a potassium phosphate buffer solution. Immediately after addition of the enzyme solution to the buffer, each of the above reagents in different concentrations was added to determine the effects of the mixtures on the enzyme activity for 6 minutes. Triethanolamine, monoethanolamine and hydrazine hydrate enhanced the enzyme activity. The largest increase, about 46%, was observed in the monoethanolamine mixture at the final concentration of 3.3×10 -2 M. The second largest increase was found in triethanolamine and the smallest in hydrazine hydrate.2) Hydroxylamine hydrochloride and ammonium sulfate inhibited the activity of glutamic acid dehydrogenase; ammonium sulfate inhibited the enzyme activity by about 50% at the final concentration of 5.0×10 -3 M, and the percentage was higher than that caused by hydroxylamine hydrochloride.