Sodium-potassium Phosphate Buffer Research Articles

Effect of phosphate buffer solutions on the reactions of glutathione with hydrogen peroxide and peroxyl radicals

Differences in the kinetics and mechanism of the reaction of glutathione (GSH) with hydrogen peroxide (H2O2) in deionized water and in phosphate buffer systems with pH ≥ 7 frequently used in biochemical studies were revealed. The formation of GSH dimers and complexes with H2O2 in water plays a substantial role in the kinetics of the process, which is manifested as nonlinear dependences of the rate of GSH consumption (WGSH) and the rate of radical formation (Wi) on the reagent concentrations. In phosphate buffer solutions (PBS), the oxidation of GSH by air oxygen is enhanced and the radical formation rate decreases sharply. An effect of NaCl and KCl in PBS on WGSH and Wi was observed, unlike a sodium—potassium phosphate buffer mixture (PB). Under other equivalent conditions, WGSH PBS is several times lower and Wi is higher than those in PB containing no chlorides. It was found that the rate of the thiol-ene reaction of unsaturated phenol resveratrol (RVT) with GSH initiated by the radicals formed in the presence of H2O2 in PBS is nearly three times lower than that in water, whereas in PB resveratrol is not consumed under the same conditions. However, in the reactions with peroxyl radicals formed upon the decomposition of 2,2′-azobis(2-methylpropionamidine) dihydrochloride the GSH consumption rate is the same in both phosphate buffer systems.

Thermal studies, degradation kinetic, equilibrium solubility, DFT, MIR, and XRPD analyses of a new cocrystal of gemfibrozil and isonicotinamide

A synthesis of a novel pharmaceutical cocrystal of the gemfibrozil (GEM) with the isonicotinamide (INCT) as coformer was carried out using the mechanochemical method of solvent drop grinding. The cocrystal formation was evidenced using the X-Ray powder diffraction, medium infrared spectroscopy, computational methods (DFT), simultaneous thermogravimetric-differential thermal analysis (TG/DTG–DTA), and differential scanning calorimetry. The association of techniques used to analyze all proportion synthetized (GEM:INCT—5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5) showed formation of the cocrystal in the proportion of 1 mol:1 mol. Furthermore, kinetic studies were performed to determine the stability of the cocrystal formed, possible shelf time (5% degradation), and best storage temperature. The dissolution profiles of GEM and GEM:INCT were collected using 0.1 M HCl, pH 4.5 sodium acetate buffer, water, and potassium phosphate buffer at pH 5.8, pH 6.8, and pH 7.4. The results demonstrate that the cocrystal exhibits superior apparent maximum solubility relative to the pure drug with a high solubility in alkaline environment. Finally, the study shows that the cocrystal of gemfibrozil with isonicotinamide can be applied in new solid oral dosage formulations of enhanced bioavailability.

Solvent and pH Dependences of Mixing Enthalpies of N-Glycylglycine with Protocatechuic Acid

Protocatechuic acid (PA) is a natural phenolic compound which has been proven to have chemopreventive property against chemically induced carcinogenesis. The mixing enthalpies of PA with N-glycylglycine in sodium phosphate and potassium phosphate buffer solutions with different pH values have been investigated by mixing-flow isothermal microcalorimetry at T = 298.15 K. The heterotactic enthalpic interaction coefficients (hxy) in the pH range of phosphate buffer solution from 3.0 to 8.0 have been calculated according to the McMillan–Mayer theory. Trends of the enthalpic pairwise interaction coefficients (hxy) with increasing pH in both phosphate buffer solutions were obtained. The solvent and pH dependence of the hxy were discussed in terms of molecular interactions between solvated solute molecules.

PH effect on the enthalpy of dilution and volumetric properties of protocatechuic acid at T = 298.15 K

The enthalpies of dilution of protocatechuic acid, a natural anti-cancer substance, in sodium phosphate and potassium phosphate buffer solutions with different pH values were measured by using a mixing-flow microcalorimeter at T=298.15K. Densities of the pseudo binary system (phosphate buffer+protocatechuic acid) were also measured with a quartz vibrating-tube densimeter. The enthalpic interaction coefficients (h2, h3 and h4) were computed according to the McMillan–Mayer model. Apparent molar volumes of the system were calculated from the data of densities, which have been used to deduce limiting partial molar volumes (Vϕ0) of protocatechuic acid at different pH values. The aim of the experiments and data process is to investigate the interaction between the molecules of the important drug and that of the drug molecule with coexistent species in aqueous solutions as well as the influences on these interactions of such factors as pH and ion strength. Change trends of the enthalpic pair wise interaction coefficient h2 and Vϕ0 of protocatechuic acid with pH increasing in the both phosphate buffer solutions were obtained. The thermodynamic properties, h2 and Vϕ0 in potassium phosphate buffer solutions were compared with those in sodium phosphate buffer solutions at identical pH. The trends and the differences have been discussed in terms of the (solute+solute) and (solute+solvent) interactions.

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

Tris–sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

Optimization of Anti-Rh D Immunoglobulin Stability in the Lyophilization Processes

Objective Anti-Rh D IgG is used for the prevention of anti-D antibody production in Rh- individuals who have been exposed to Rh+ red blood cells. The stability of IgG preparations as a solution is low, with a shelf life of a year or more. Formulation of anti-Rh D IgG as a lyophilized preparation would decrease its degradation rate and increases its shelf life. The objective of this study was to formulate the anti-Rh D as a lyophilized preparation using different formulations and optimize the lyophilization processes. Materials and Methods The effect of various formulations on the stability of anti-Rh D was evaluated using accelerated stability test. In this method the amount of transmittance (T %) at 585 nm for the lyophilized preparations had inverse relationship with aggregation of anti-Rh D. To improve stability, the most stable formulation was selected and different concentrations of sucrose in the presence of sodium-potassium phosphate buffer 25 mM pH 7.5. Then, the bioactivity was determined, using the ELAT test and also, the amount of moisture measured in this formulation. Results Among different formulations, the one with anti-Rh D 5 mg/ml, tween 80 0.1%, glycine 0.15 M, manitol 7% and sucrose 60 mM in sodium-potassium phosphate buffer 25 mM pH 7.5 was the most stable formulation (P<0.05). The result of biological test of ELAT showed that bioactivity of more than 93% meets the requirement set by British Pharmacopoeia. The amount of moisture measured in this formulation was less than 3%. Conclusion It was concluded that this formulation could be introduced as a candidate for the formulation of anti-Rh D in a lyophilized dosage form.

Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (R)-limonene in organic media

Horse radish peroxidase (HRP) from Armoracia rusticana catalyses the oxidation of ( R )-limonene into the oxidized derivatives carveol and carvone. This study compares the direct addition (DA) of hydrogen peroxide with its continuous electrogeneration (EG) during the enzymatic oxidation of ( R )-limonene. Reaction mixtures containing HRP, ( R )-limonene as substrate, and hydrogen peroxide, added directly or electrogenerated, in 100 mM sodium-potassium phosphate buffer pH 7.0, at 25oC were studied. Two electrochemical systems for the hydrogen peroxide electrogeneration were evaluated, both containing as auxiliary electrode (AE) a platinum wire and saturated calomel electrode (SCE) as reference. Reticulated vitreous carbon foam (RVCF) and an electrolytic copper web (CW) were evaluated as working electrodes (WE). Results were compared in terms of hydrogen peroxide electrogeneration, ( R )-limonene residual concentration or conversion and product selectivity. Best results in terms of maximum H 2 O 2 concentration (1.2 mM) were obtained using the CW electrode at -620 mV SCE , and continuous aeration. Use of the EG system under optimized conditions, which included the use of acetone (30% v/v) as a cosolvent in a 3 hrs enzymatic reaction, lead to a 45% conversion of ( R )-limonene into carveol and carvone (2:1). In comparison to the results obtained with DA, the use of EG also improved the half-life of the enzyme.

Simultaneous quantification of voriconazole and posaconazole in human plasma by high-performance liquid chromatography with ultra-violet detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method with ultra-violet detection has been developed and validated for the simultaneous determination of posaconazole and voriconazole, two systemic anti-fungal agents. An internal standard diazepam was added to 100 μL of human plasma followed by 3 mL of hexane–methylene chloride (70:30, v/v). The organic layer was evaporated to dryness and the residue was reconstituted with 100 μL of mobile phase before being injected in the chromatographic system. The compounds were separated on a C8 column using sodium potassium phosphate buffer (0.04 M, pH 6.0): acetonitrile:ultrapure water (45:52.5:2.5, v/v/v) as mobile phase. All compounds were detected at a wavelength of 255 nm. The assay was linear and validated over the range 0.2–10.0 mg/L for voriconazole and 0.05–10.0 mg/L for posaconazole. The biases were comprised between −3 and 5% for voriconazole and −2 and 8% for posaconazole. The intra- and inter-day precisions of the method were lower than 8% for the routine quality control (QC). The mean recovery was 98% for voriconazole and 108% for posaconazole. This method provides a useful tool for therapeutic drug monitoring.

Chloroperoxidase mediated oxidation of chlorinated phenols using electrogenerated hydrogen peroxide

Chloroperoxidase (CPO) from Caldariomyces fumago catalyses the oxidation of several chlorinated phenols (CP) commonly found in industrial waste waters in the presence of hydrogen peroxide. This study compares the direct addition of hydrogen peroxide (DA) with its continuous electrogeneration (EG) during the enzymatic oxidation of CP. Reaction mixtures were studied containing chemically modified CPO, hydrogen peroxide and the phenolic substrates: phenol (P), 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP), in 100 mM sodium-potassium phosphate buffer pH 6.0, at 25oC. Results were compared in terms of residual phenol concentration (oxidation efficiency), precipitate formation (removal) and residual enzyme activity (stability). With the electrochemical system evaluated at -620 mVSCE, and continuous aeration the maximum H2O2 concentration of 1.2 mM was obtained. Under this conditions and after 4 hrs using EG, no phenol or 4-CP were detected, and 97%, 93% and 88% of 2,4-DCP, 2,4,6-TCP and PCP were degraded, respectively. The use of EG improves enzyme half-life time in comparison to the results obtained by DA

Plasminogen activator inhibitor 1 (PAI-1) in its active conformation: crystallization and preliminary X-ray diffraction data.

Because of its intrinsic lability, wild-type plasminogen activator inhibitor 1 (PAI-1) cannot be crystallized in its active conformation. Therefore, a stable variant of PAI-1 was used to retain the active conformation during crystallization. Four different crystallization conditions were evaluated in detail and two major types of crystals were detected. Whereas solutions consisting of either (i) cacodylate and sodium acetate, (ii) lithium sulfate and polyethylene glycol 4K, or (iii) imidazole, sodium chloride and sodium potassium phosphate buffer revealed thin platelet crystals, a solution (iv) containing ammonium acetate, citrate and polyethylene glycol 4K appeared to enhance the formation of clustered brush-like crystals. Crystals grown under condition (iii) were found to be suitable for X-ray data collection and consequent structural investigation. Data collection was 79.8% complete with a maximum resolution of 2.92 A. Importantly, PAI-1 retained its functional properties under all conditions.

Sucrase activity and its kinetic properties in peritrophic membrane, and in membrane-bound and soluble fractions of midgut in the silkworm, Bombyx mori

The contribution of sucrase in the peritrophic membrane to the hydrolysis of sucrose in the midgut of the silkworm, Bombyx mori was evaluated. Sucrase activities per individual insect and its kinetic properties were determined in the peritrophic membrane and membrane-bound and soluble fractions of midgut between days 2 and 6 in the fifth instar. The activity of the sucrase in the peritrophic membrane relative to membrane-bound sucrase of midgut increased, from day 2 to day 6, approximately 5-fold; from 1:334 to 1:71 determined in sodiumpotassium-phosphate buffer, which appeared to indicate an increasing significance of the sucrase in the peritrophic membrane during this developmental period. The sucrase hi the peritrophic membrane showed high activity at alkaline pH, 8.0–11.0 as well as at pH 6.5–8.0. A shift towards higher activity was observed from day 4 to day 6, at pH 5.0–6.5 in all three sucrases, but at pH 8.0–11.0, a smaller shift was observed in the sucrase in the peritrophic membrane. The Km of the sucrase in the peritrophic membrane was 1.2 mM and an apparent substrate activation was observed, which was similar to the characteristics of the membrane-bound sucrase of midgut. Determination of sucrase activity in the presence of various metal ions at 1 and 10 mM disclosed that most of the metal ions exerted inhibitory effects, notably in Hg2+, Cu2+ and Zn2+. Similar effects were observed with the sucrase in the peritrophic membrane and soluble sucrase of midgut. The effects of Tris and Tricine, and phlorizin, phloretin and ouabain on the activity of the membrane-bound sucrase were investigated. Among them, Tris was the most potent inhibitor followed by Tricine; 40 and 30% of the control activity were observed at final 32 and 150 mM, respectively. Phlorizin and ouabain exerted no effect, whereas Phloretin caused the activity to reduce to 80% of the control at the final 400 mM.

Cryoprotection of purified rat kidney transamidinase by polyethylene glycol

Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 m M ethyl-enediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.

Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells

We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl- l-methionine and magnesium chloride in sodium—potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl- l-methionine using this method are similar to values reported previously (0.51 and 14 μ M, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2–3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.

A colorimetric assay for measuring cell‐free and cell‐bound cholesterol oxidase

Cholesterol oxidase (cholesterol:oxygen oxidoreductase, EC 1.1.3.6) catalyzes the conversion of sterol delta 5-3 beta-alcohol to the corresponding delta 4-3-ketone with the reduction of oxygen to hydrogen peroxide. Rhodococcus species GK 1, a soil isolated microbe, produces an extracellular and a membrane-bound cholesterol oxidase; the latter is bound to the outer surface of the microbial cell membrane. A simple and sensitive assay is described to measure the two enzyme types; no enzyme extraction is needed for measuring the membrane-bound cholesterol oxidase. In this assay, hydrogen peroxide is reduced by the chromogen 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of horseradish peroxidase, and the increased absorbance is followed continuously at 600 nm (epsilon m = 1.82 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C). The standardized assay medium contained 46.9 mM sodium-potassium phosphate buffer pH 7.0, 0.16% Triton X-100, 312.5 microM ABTS, 50 micrograms peroxidase (12.5 units at 25 degrees C), 6.25% isopropanol, 306.3 microM cholesterol or other sterols (kept in solution with isopropanol), and cholesterol oxidase. Oxidation of one molecule of cholesterol by cholesterol oxidase gives one molecule of hydrogen peroxide which reacts with two molecules of ABTS. The method is reproducible and the results correlate well with those obtained by measuring the absorbance of delta 4-cholest-3-one at 240 nm (epsilon m = 1.40 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C) and by the method of Allain et al. (Clin. Chem. 20, 470-475, 1974).(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobin aggregation and pseudosickling in vitro of hemoglobin setif-containing erythrocytes

Erythrocytes from individuals heterozygous for hemoglobin Setif (alpha 94 Asp----Tyr) sickle in vitro without deoxygenation when incubated in chloride buffer due to hemoglobin aggregation. We now report quantitative studies of hemoglobin polymerization and deformability in these cells. Hemoglobin polymer gradually increased in intact cells during a 24 h incubation period at 24 degrees C. After 24 hr, about 80% of the cells in 290 mOsm sodium chloride buffer contained polymer which appeared as short rods compared to greater than 99% containing polymer at 450 mOsm. Similar proportions of cells were morphologically sickled. Deformability of erythrocytes with 40% hemoglobin Setif incubated in 290 mOsm buffer at 37 degrees C decreased to 80% of normal by 210 min but in 450 mOsm decreased to 50% after only 30 min as measured by the ektacytometer. However, at 4 degrees C deformability remained normal even in 450 mOsm buffer. The solubility of gelled hemolysate containing 40% hemoglobin Setif was 24 g/dl and 21 g/dl at 290 and 459 mOsm buffer respectively. The gel persisted at 4 degrees C with a solubility of 26 g/dl, but melted when dialyzed into sodium phosphate or potassium phosphate buffer. These data suggest that hemoglobin polymerization, reduced deformability, and sickling of hemoglobin Setif-containing erythrocytes are related to reduced hemoglobin solubility. The rate and extent of intracellular polymerization in vitro are considerably reduced (as in the case of sickle trait) compared with erythrocytes from individuals with sickle cell anemia. Hence, the slower kinetics of hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin polymerization and abnormal rheology.

Immunogold labeling of phycobilisomes in a cyanobacterium

Phycobilisomes are pigment-protein complexes that act as the major light- harvesting antenna, in addition to chlorophyll, for photosynthesis in red algae and Cyanobacteria.In vivo, phycobilisomes transfer their energy mainly to the photosystem II reaction center to which they are assumed to be attached. This assumption will be tested by the use of gold labeling of antibodies raised against photosystem II reaction center components to localize the reaction centers and to compare the location of the antibodies with that of the phycobili- somes. In Cyanobacteria phycobi1isomes are difficult to fix particularly under conditions required for immunocytochemistry. Here, two methods for efficient fixation of phycobi1isomes are described, together with immunocytochemical results to confirm the identity of the phycobilisomes.For phycobilisome detection,Synechocvstissp. PCC 6803 cells were either freeze substituted or fixed in a high molarity buffer followed by cryomicrotomy. For freeze substitution the cells were collected, subjected to ultra-rapid freezing with a slammer device and a copper block at liquid helium temperature, placed in 5% acrolein in ethanol at -85°C for 4 days, and embedded in LR White. For cryo- sectioning the cells were fixed with 1% glutaraldehyde in 0.75M sodium potassium phosphate buffer, pH 7.4, for 2 h, enrobed in 10% gelatin, infused with 1.6M sucrose in 0.1M sodium phosphate buffer,pH 7.2, for 3 h, and frozen in liquid freon. Cryosections were prepared with an RMC CR2000 cryoultramicrotomy unit on an MT-6000 ultramicrotome. In both procedures 0.05% Carnation nonfat dried milk in 0.1M sodium phosphate buffer was used to block nonspecific binding of the anti serum. Sections were treated with rod-phycocyanin (phycobilin protein) anti - serum followed by protein A conjugated to lOnm gold particles. Subsequently the plastic sections were stained with uranyl acetate and lead citrate. The cryosections were subsequently treated with 1% OSO4, 1% tannic acid, uranyl acetate, and alkaline bismuth, dehydrated in ethanol, and embedded in LR White. Sections were observed in a Philips EM201.

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.

Assay of superoxide dismutase activity in animal tissues

Convenient assays for superoxide dismutase have necessarily been of the indirect type. It was observed that among the different methods used for the assay of superoxide dismutase in rat liver homogenate, namely the xanthine-xanthine oxidase ferricytochromec, xanthine-xanthine oxidase nitroblue tetrazolium, and pyrogallol autoxidation methods, a modified pyrogallol autoxidation method appeared to be simple, rapid and reproducible. The xanthine-xanthine oxidase ferricytochromec method was applicable only to dialysed crude tissue homogenates. The xanthine-xanthine oxidase nitroblue tetrazolium method, either with sodium carbonate solution, pH 10.2, or potassium phosphate buffer, pH 7·8, was not applicable to rat liver homogenate even after extensive dialysis. Using the modified pyrogallol autoxidation method, data have been obtained for superoxide dismutase activity in different tissues of rat. The effect of age, including neonatal and postnatal development on the activity, as well as activity in normal and cancerous human tissues were also studied. The pyrogallol method has also been used for the assay of iron-containing superoxide dismutase inEscherichia coli and for the identification of superoxide dismutase on polyacrylamide gels after electrophoresis.

Inhibition of mechanical transmission of virus by sap, and infection of Cucurbita ecuadorensis with papaya ringspot virus type W

Dilutions of Cucurbita ecuadorensis sap were mixed with dilutions of sap from C. maxima infected with papaya ringspot virus type W (PRV-W), and the mixtures mechanically inoculated to seedlings of C. pepo cv. Small Sugar or Cucumis sativus cv. Supermarket. Plants inoculated with C. maxima sap plus C. ecuadorensis sap (at 1/8 when C. pepo was the test plant, or 1/16 when C. sativus was the test plant) showed reduced infection compared with plants not receiving C. ecuadorensis sap. The reduction caused by C. ecuadorensis sap was greater at a lower virus concentration (1 g infected C. maxima/400 mL sodium-potassium phosphate buffer 0.1 mol/L) than at a higher virus concentration (1 g/25 mL). It was concluded that C. ecuadorensis sap contains an inhibitor of infection, that tissue derived from this species should be diluted at least 1 g/ 10 mL buffer before use in inoculations, and that C. pepo, which was 5 times more sensitive than C. sativus, should be used as the test seedling in reisolations. When C. ecuadorensis, C. maxima and the F1 (C. maxima x C. ecuadorensis) were inoculated on their cotyledons with a severe isolate of PRV-W, symptoms were expressed on at least 1 plant of each species and the F1, and the virus was reisolated in seedlings of C. pepo cv. Small Sugar from the uninoculated leaves of all plants. Severity of symptoms and the proportion of plants infected at reisolation were least for C. ecuadorensis, intermediate for the F1 and most for C. maxima. This is the first report of infection of C. ecuadorensis by PRV-W.