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Glutaminases
Published in Elling Kvamme, Glutamine and Glutamate in Mammals, 1988
Elling Kvamme, Gerd Svenneby, Ingeborg Aasland Torgner
A different type of activator sensitizes PAG to activation by phosphate. This is exemplified by the effect of the dye Bromothymol Blue on pig kidney and brain PAG. In low concentrations the dye enhances the activation by phosphate, whereas it inhibits the enzyme activity at higher concentrations,6,39,40 but phosphate protects against this inhibition.40 The effects of Bromothymol Blue mimic similar effects by physiological compounds, such as acyl-CoA derivatives,40-42 calcium,35,43-45,82 and thyroxine.46-48
Ultraviolet and Light Absorption Spectrometry
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Zoltan M. Dinya, Ferenc J. Sztaricskai
Sanghavi and Katdare [218] first hydrolyzed erythromycin with concentrated hydrochloric acid followed by treatment with benzaldehyde in acetic acid to obtain a Schiff base exhibiting absorption at 490 nm. In these quantitative measurements another antibiotic, such as penicillin, neomycin, or streptomycin, did not interfere, but the presence of tetracyclines disturbs the determinations. Later, benzaldehyde was replaced by p-dimethylaminobenzaldehyde and the measurements were achieved at 488 nm [219]. Amer et al. [220] estimated the erythromycin content of pharmaceutical preparations on the basis of the absorbance at 386 nm of a yellow product formed on treatment with salicylaldehyde in ethanol. Smith et al. [221] elaborated a colorimetric assay method for the chloroform-extractable complexes of erythromycin with sulfonic acid dyes (such as methyl orange). It has been shown by Bhathar and Madkaiker [200] that erythromycin also forms a complex with bromothymol blue at pH 3.5, which is extractable with chloroform and exhibits specific absorbance at 415 nm. Among several sulfophthalein acid dyes, bromophenol blue was applied by Shirokova and Charykov [222] for the determination of erythromycin. It has been established by Regosz et al. [223] that a pH 4.2 medium is optimal for the use of bromophenol blue and the absorbance at 415 nm is suitable for the assay of erythromycin content of pharmaceutical preparations. By applying bromothymol blue, an analytical method was elaborated by Slavin et al. [224] for the estimation of oleandomycin.
Glycerine Analysis
Published in Eric Jungermann, Norman O.V. Sonntag, Glycerine, 2018
In general, the analysis is performed in the following way [8]: A quantity of sample containing between 0.32 (3.47 mmol) and 0.5 (5.42 mmol) of glycerine is dissolved in 100–150 ml of distilled water. Accurate estimation of the expected glycerine level is important. The method becomes inaccurate if too much glycerine is present, since an excess of periodate is required for quantitative reaction. Similarly, if too little glycerine is present, the final titration of glycerine will take too little standardized base for accurate reading of the burret.The solution is carefully neutralized to a pH of 8.1 with a pH meter or an indicator (e.g., bromthymol blue). This is required because it corresponds to the neutralization point of the solution after the oxidation reaction.A 50-ml aliquot of 0.28 M sodium metaperiodate (14 mmol) is added to the solution, and it is reacted at room temperature for about 30 minutes. Since each millimole of glycerine requires 2 mmol of periodate for reaction, these conditions are sufficient to assure an excess of periodate, even when 0.5 gram (5.42 mmol) is present.After this 30 minute reaction period, excess ethylene glycol is added to react with the periodate. The ethylene glycol is oxidized to formaldehyde.The formic acid produced is determined by titration with standardized sodium hydroxide to a pH of 6.5. This lower pH endpoint is used because it corresponds to the equivalence point of the formic acid/formate titration.
Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Mariantonia Logozzi, Davide Mizzoni, Clemente Capasso, Sonia Del Prete, Rossella Di Raimo, Mario Falchi, Daniela F. Angelini, Alessandro Sciarra, Martina Maggi, Claudiu T. Supuran, Stefano Fais
Exosomes were obtained from plasma of 8 prostate cancer patients (PCa) and 8 healthy donors (CTR). Exosome extracts were prepared at 4 °C using the lysis buffer (CHAPS buffer 1x) containing 1% Triton X-100, 10mMTris-HCl (pH 7.4), MgCl2 1 mM, EGTA 1 mM, CHAPS 0.5%, glycerol 10%, β-mercaptoethanol 5 mM, and supplemented with a cocktail of protease inhibitors. Aliquots of exosomes extracts containing 1 µg of total protein were used to determining the hydratase activity. The enzymatic assay was performed at 0 °C using CO2 as substrate following the pH variation due to the catalysed conversion of CO2 to bicarbonate. Bromothymol blue was used as the indicator of pH variation. The production of hydrogen ions during the CO2 hydration reaction lowers the pH of the solution until the colour transition point of the dye is reached. The time required for the colour change is inversely related to the quantity and activity of CAs present in the sample. Wilbur–Anderson units were calculated according to the following definition: One Wilbur–Anderson unit (WAU) of activity is defined as (T0 − T)/T, where T0 (uncatalyzed reaction) and T (catalysed reaction) are recorded as the time (in seconds) required for the pH to drop from 8.3 to the transition point of the dye (pH 6.8) in a control buffer and in the presence of enzyme, respectively. Enzyme activity was expressed as CA activity/mg of total protein. Protein concentration was determined using the Bio-Rad protein assay.
Use of an immobilised thermostable α-CA (SspCA) for enhancing the metabolic efficiency of the freshwater green microalga Chlorella sorokiniana
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Giovanna Salbitani, Sonia Del Prete, Francesco Bolinesi, Olga Mangoni, Viviana De Luca, Vincenzo Carginale, William A. Donald, Claudiu T. Supuran, Simona Carfagna, Clemente Capasso
CA activity assay was performed as described by Capasso et al.62. Briefly, the assay was based on the monitoring of pH variation due to the catalysed conversion of CO2 to bicarbonate. Bromothymol blue was used as the indicator of pH variation and the assay was performed at 0 °C. The CO2-satured solution was used as substrate. To test the activity of carbonic anhydrase, 1.0 mL of 25 mM Tris, pH 8.3, containing bromothymol blue as a dye (to give a distinct and visible blue colour) was added to two test tubes chilled in an ice bath. An appropriate amount of the enzyme solution (e.g. microalgal cell extract) were added to one tube, and an equivalent amount of buffer was added to the second tube as control. One millilitre of CO2 solution was added, and the time required for the solution to change from blue to yellow was recorded (transition point of bromothymol blue is pH 6.0–7.6). The time required for the colour change is inversely related to the quantity of enzyme present in the sample. Wilbur-Anderson units were calculated according to the following definition: One Wilbur-Anderson unit (WAU) of activity is defined as (T0−T)/T, where T0 (uncatalyzed reaction) and T (catalysed reaction) are recorded as the time (in seconds) required for the pH to drop from 8.3 to the transition point of the dye in a control buffer and in the presence of enzyme, respectively.
Thermostability enhancement of the α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-self-labelling-protein-tag system (ASLtag)
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Sonia Del Prete, Rosa Merlo, Anna Valenti, Rosanna Mattossovich, Mosè Rossi, Vincenzo Carginale, Claudiu T. Supuran, Giuseppe Perugino, Clemente Capasso
CA activity assay was a modification of the procedure described by Capasso et al.59. Briefly, the assay was performed at 0 °C using CO2 as substrate and following the pH variation due to the catalysed conversion of CO2 to bicarbonate. Bromothymol blue was used as the indicator of pH variation. The production of hydrogen ions during the CO2 hydration reaction lowers the pH of the solution until the colour transition point of the dye is reached. The time required for the colour change is inversely related to the quantity of CA present in the sample. Wilbur-Anderson units (WAU) were calculated according to the following definition: one WAU of activity is defined as (T0−T)/T, where T0 (uncatalysed reaction) and T (catalysed reaction) are recorded as the time (in seconds) required for the pH to drop from 8.3 to the transition point of the dye in a control buffer and in the presence of enzyme, respectively. Assay of the membrane-bound enzyme (H5-SspCA or SspCA) was carried out using an amount of whole cells or outer membranes ranging from 1.0 to 5.0 mg. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli using 12% gels.60 Samples were dissolved in buffer with 5% β-mercaptoethanol. The gel was stained with Coomassie blue and protein concentration was determined by Bio-Rad assay kit (Bio-Rad, Hercules, CA).