Phenol Red as pH Indicator

This assay principle has been successfully developed also to monitor enzymatic reactions that involve various types of ester [9,29,30] and hydantoin hydrolysis [30a], phosphoryl transfer [31], nucleotidyl transfer [32] and glycosyltransfer [33, 34], as well as decarboxylation reactions [35]. The advantages of pH-based assay methods are obvious pH indicators are inexpensive reagents, no auxiliary enzymes are required, initial reaction rates can be monitored continuously in real time, and the reaction principle should be easily adaptable for HTS in microtiter plate format. 

Using phenol red as a sensitive pH indicator in the presence of low buffer concentration (2 niM triethanolamine (TEA), pH 7.5), an assay has been developed for the reliable colorimetric determination of TK activity. This new continuous, generic pH-based method proved suitable for the quantitative determination of kinetic parameters, for individual substrates, for the rapid mapping of an enzyme s substrate tolerance, as well as for the HTS of TK variants. 

Using this assay, the substrate specificities of the TK enzymes from E. coli and S. cerevisiae, as well as two active site modified variants (D469E, H26Y) of E. coli TK were evaluated against a panel of natural and nonnatural substrate analogs, such as hydroxylated and nonhydroxylated, nonphosphorylated acceptor substrates, for which specific activities and kinetic constants were determined. 

The assay solution contained 50 or 200 mM acceptor, 4,12, or 32 pg TK, 2.4 mM TPP, 9mM MgCl2, 0.028 mM phenol red, and 2mM TEA (pH 7.5). TTie reaction started after adding 50 mM LiHPA, Total assay volume was 200 pi. The absorbance increase was measured at 560 nm by plate reader. 

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The binding capacity obtained with this material was between 18 and 28 mg of IgG per milliliter of silica as matrix. When agarose beads were used, binding capacity was of about 15 mg/mL. It has been, however, indicated that with cell culture supernatants containing phenol red as pH indicator, binding capacity decreased to few milligrams of IgG per milliliter of resin. 

The solution is decanted from the mercury and filtered from suspended impurities. The filtrate is stirred and is acidified with hydrochloric acid to pH 6.6, using phenol red as the indicator (Note 3). After standing for two hours at 0°, the mixture is filtered and the solid is washed with a little cold water and dried. The product (free base) is microcrystalline, weighs 17.5-18 g. (72-74 per cent of the theoretical amount), and usually melts at 167-169° (Note 4). After solidification from fusion, the substance melts at 226-228°. When recrystallized from water, the substance melts at 231-233°. The crude product may be used for the subsequent hydrolysis. 

The first pH sensor was developed at NIH (Bethesda, Maryland) and made use of phenol red as acid-base indicator, covalently bound to polyacrylamide microspheres10 such microspheres are contained inside a cellulose dialysis tubing (internal diameter 0.3 mm) connected to a 250 pm plastic fibre (Figure 2). The probe was inserted into either the tissue or the... 

Hanks Solution. For tissue culture (sterile, phenol red added as pH (indicator) (Sigma H8389). This replaces the more expensive MEM (minimal essential medium). 

A wide variety of pH indicators are availaUe from analytical chonistry. Since indicators are intended for visual observation, they display pH-dependent absorption spectra, with absmption at visible wavdengths. These indicators have formed the basis for a number of RET pH/jpCOz sensors. One of the earliest reports used eosin as the donor and Phenol Red as the acceptor. Phenol Red was selected becauseit displays apiK. near 7, and the basic form absorbs at S46 nm, when eosin emits. Consequently, the eosin intensity decreased as the pH increased. In the case of this particular sensor, it was not certain whether the decreased intensity was due to RET or to an inno- filto- effect, but it is dear that RET is a usdiil mechanism as a basis for designing sensors. 

Kinetics of Proton Uptake. The Rinetics of proton uptaRe were measured for both native and mutant RCs using the pH indicator dye phenol red as described in Sec. 3. In RCs of Rb. sphaeroides R-26, 2.0 H /Qb" were taRen up within the experimental time resolution (Fig. 

Optical fiber detectors are usually utilized as ether miniature spectrophotometers or fluorimeters, because of the availability of indicator dyes that can be detected by absorbance or fluorescence methods. Figure 12 shows the absorption spectrum for phenol red, a dye commonly used as pH indicator. By measuring the absorption of solutions of phenol red at two wavelengths, the isobestic point where absorption is virtually independent of pH and at 600 m u where the intensity of absorption is very pH sensitive, one... 

Schmidt and coworkers have carried out determinations of the surface acidity of pharmaceutical excipients and used a variety of absorption indicators to span the ranges of observed the acid strengths. In one study, the surface acidity of microcrystalline cellulose and dicalcium phosphate anhydrate was evaluated using thymol blue, bromcresol green, bromcresol purple, and phenol red as the absorption indicators. " Using the pH dependence of dye absorbance published in the paper, it was concluded that Hq was approximately 1.0 for dicalcium phosphate anhydrate, and approximately 4.0 for microcrystalline cellulose. 

Adjust the penicillinase to pH 7 5 using phenol red as indicator. Prepare a colour control by mixing 1 ml of this with 10 ml of distilled water containing 0 2 ml of phenol red indicator. Weigh accurately 50 mg of the penicillin sample, dissolve in 10 ml of water also containing 0-2 ml of phenol red indicator, and adjust the pH value to match that of the control. Add 1 ml of penicillinase and allow to stand at room temperature for thirty minutes. Titrate with 0-01N sodium hydroxide until the colour of the solution again matches that of the control. Allow to stand for some minutes longer to ensure that the reaction is completed, and titrate further if necessary. Calculate the potency of the preparation on the basis that 1 ml of O OIK sodium hydroxide is equivalent to 6023 I.U. of p( nicillin. 

The principle of this test is as follows The liquid suspected of containing urea is treated with dilute acid or alkali until its pH is about 7. A solution of the enzyme is also made and its pH adjusted to 7. The two solutions are mixed and the resulting conversion of urea to ammonium carbonate causes the pH of the solution to rise to over 8 this change is noted by the use of a suitable indicator, phenol-red being the one usually employed. Proteins do not interfere with the test, but the reaction is inhibited by traces of heavy metals. 

With 0.01M solutions, the ideal pH range is still further limited to 5.5-8.5 such indicators as methyl red, bromothymol blue, or phenol red will be suitable. The titration error for methyl orange will be 1-2 per cent. 

Gravimetric and volumetric methods are practicable for the quantitative determination of the a-sulfo fatty acid esters. Using gravimetric methods the surfactant is precipitated with p-toluidine or barium chloride [105]. The volumetric determination method is two-phase titration. In this technique different titrants and indicators are used. For the analysis of a-sulfo fatty acid esters the quaternary ammonium surfactant hyamine 1622 (p,f-octylphenoxyethyldimethyl-ammonium chloride) is used as the titrant [106]. The indicator depends on the pH value of the titration solution. Titration with a phenol red indicator is carried out at a pH of 9, methylene blue is used in acid medium [106], and a mixed indicator of a cationic (dimidium bromide) and an anionic (disulfine blue VN150) dye can be used in an acid and basic medium [105]. 

The pH change can be monitored using a colorimetric pH indicator dye such as chlorophenol red,22 phenol red or bromothymol blue41. 

Reduction of D-proline by D-amino acid oxidase at pH 8 shows two steps when monitored at 640 nm. These are interpreted as the build-up and breakdown of a reduced enzyme-imino acid charge transfer complex. If the reaction is monitored using phenol red the same two rates are observed but additionally the release of = 1 proton for each step can be assessed and interpreted. The indicator changes are followed at 505 nm and 385 nm, which are isosbestic wavelengths for the two steps (without indicator),... 

Recently, DeGrandpre [12] developed a probe-type sensor for the determination of PCO2 in sea water by direct immersion of the probe, which, however, has some connotations of flow-through sensor even though a pH indicator such as Phenol Red (piTj = 7.5) or Bromothymol Blue (pAn = 6.8) rather than the sample is circulated over the sensing microzone —the basic forms of these indicators have a high molar extinction coefficient at 560 and... 

Solutions of indicators are out for pH measuring, but fhey are useful for monitoring some processes, such as tissue cultures (indicator phenol red) or electrophoresis (electrophoresis front and pH of sample indicator bromophenol blue). 

A study of phenol red binding under different pH conditions may be completed by changing the pH of the reaction mixtures and Sephadex gel column. For each pH to be studied, the column must first be equilibrated with the proper buffer. Several buffers are available including acetate buffer (pH 4.0, 4.5, and 5.0) and phosphate buffer (pH 6.0, 7.0, and 8.0). Equilibrate the column with approximately 20-25 mL of the new buffer. To be assured of the proper pH, check the column eluent with a pH indicator strip or collect a fraction for measurement with a pH meter. Prepare the reaction mixtures by mixing the protein with the appropriate buffer and phenol red. Be sure to note that solutions of phenol red are prepared in different buffers. Load the reaction mixture on the equilibrated Sephadex G-25 column and develop and analyze the column fractions as described above. 

Ping et al. have fabricated an integrated microsensor array on a silicon wafer for pH imaging [89]. Six different pH-sensitive colorimetric dyes (methyl violet 6B, phenolic red, alizarin complexone, 5-carboxy-fluorescein, alizarin red and methylthymol blue) were used to cover the whole pH range. The dyes were adsorbed on microbeads and placed in etched microwells on the silicon wafer. The indicator array was also used as a cation sensor chip (see Sect. 2.4). 

Do not put a pH probe into the HBSS mixtures to be used for eosinophil purification and labeling. Adjust the pH of HBSS/BSA and HBSS/citrated plasma by adding 0.1 MNaOH until the color of the phenol red indicator in the HBSS shows pH 7.3. (Remove an aliquot to check the pH and then discard.) Adjust the pH of Percoll/HBSS by cautious addition of 1M HC1 with mixing to prevent formation of a precipitate. Use the color of the phenol red indicator as a guide, but only remove a small aliquot for pH measurement if the pH probe will function with 100 pL or less (and then discard). 

The assay sample buffer we have used is 0.14 M sodium chloride with 10 mM phosphate, pH 7.0, phenol red at 10 mg/liter, and an inert protein, usually 0.1% gelatin. The protein is included in all samples to minimize adsorptive losses. Gelatin has proved to be most useful because it is free of most potentially cross-reactive proteins that occasionally contaminate some preparations (e.g., luteinizing hormone in crystalline bovine serum albumin) it is free of most small, nonproteinaceous molecules that occasionally contaminate other preparations (e.g., steroids in ovalbumin) it effectively reduces nonspecific adsorption it is inexpensive and it does not cause foaming or create problems with valves on some automatic pipetting equipment. The concentration of phosphate is low and could be increased or supplemented. In effect we accomplish this by including 50 mM EDTA in the buffer used for the first antibody. The phenol red is included to serve as an indicator of dangerous pH shifts upon addition of sample. 

These acid- or alkaline-balanced products may be advertised as pH balanced. But what does pH mean We can get a clue from revisiting the pool test kit. The phenol red indicator is also labeled pH indicator pH is a measure of how acidic or basic a solution is. It is a measure of the acid quality of a solution. 

The pH indicator phenol red (phenol sulfonphtha-lein) is often added to commercially available media. It is pale yellow at pH 6.5, orange at pH 7.0, and red at pH 7.4. It becomes purplish above pH 7.4. Growth media are also often supplemented with antibiotics to promote the growth and propagation of antibiotic resistant strains and also to prevent contamination by micro-organisms. But the presence of antibiotics in the medium does not obviate the use of good aseptic techniques. Nowadays, however, most commercially available media are presterilized as are the polystyrene culture dishes. 

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