Glutaminases
Elling Kvamme in Glutamine and Glutamate in Mammals, 1988
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
Adorjan Aszalos in Modern Analysis of Antibiotics, 2020
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.
Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of sulphonamide carbonic anhydrase inhibitors
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Alessandra Rogato, Sonia Del Prete, Alessio Nocentini, Vincenzo Carginale, Claudiu T. Supuran, Clemente Capasso
CA activity assay was a modification of the procedure described by Capasso et al.57. Briefly, the hydratase 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 pH indicator. The production of hydrogen ions during the CO2 hydration reaction lowers the pH of the solution leading to a colour transition of the dye. The time required for the colour change is inversely proportional to the amount of CA present in the sample. The Wilbur-Anderson units (WAU) were calculated according to the following definition: one WAU of CA activity is defined as the ratio (T0 − T)/T, where T0 (the time needed for the pH indicator colour change for the uncatalysed reaction) and T (the time needed for the pH indicator colour change for the 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 the presence of enzyme, respectively.
An AGT-based protein-tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Rosa Merlo, Sonia Del Prete, Anna Valenti, Rosanna Mattossovich, Vincenzo Carginale, Claudiu T. Supuran, Clemente Capasso, Giuseppe Perugino
CA activity assay was a modification of the procedure described by Capasso et al.33. Briefly, the hydratase assay was performed at 0 °C using CO2 as substrate following the pH variation due to the catalyzed conversion of CO2 to bicarbonate. Bromothymol blue was used as pH indicator. The production of hydrogen ions during the CO2 hydration reaction lowers the pH of the solution leading to a colour transition of the dye. The time required for the colour change is inversely proportional to the amount of CA present in the sample. The Wilbur–Anderson units (WAU) were calculated according to the following definition: one WAU of CA activity is defined as the ratio (T0 − T)/T, where T0 (the time needed for the pH indicator color change for the uncatalyzed reaction) and T (the time needed for the pH indicator color change for the catalyzed 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 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).
Related Knowledge Centers
- Benzene
- Bromocresol Green
- Carbonic Acid
- Diethyl Ether
- Sodium
- Toluene
- Ph Indicator
- Salt
- Chlorophenol Red
- Thymol Blue