Binding of phenol red

For each reaction mixture, calculate the bound phenol red as the percentage of total phenol red as above. Prepare a plot of % bound (y-axis) versus pH. In previous studies it has been shown that optimum binding of phenol red to BSA occurs in the pH range of 3 to 5. Binding affinity gradually declines between pH 5.0 and 8.0 and is insignificant above pH 8. 

Below is a table showing the °/o binding of phenol red to human serum albumin versus pH of the reaction mixture. Draw a graph showing the results and explain the shape of the curve. 

U. Kragh-Hansen, Biochem /. 195, 603-613 (1981). Effects of Aliphatic Fatty Acids on the Binding of Phenol Red to Human Serum Albumin. ... 

R17. Rodkey, F. L., Binding of phenol red by human serum albumin. Arch. Biochem. Biophys. 94, 526-531 (1961). 

At the dose most commonly employed in the present work (10 mg/kg), 66.6% of phenol red occurred bound as determined by ultrafiltration techniques (8). There were no significant differences in percentage bound at 20 and 40 mg/kg doses whereas there was a slight but significant decrease in the amount of binding by phenol red in the presence of probenecid. 

Extensive research on albumin has led to an increasingly clear picture of ligand binding. The dye phenol red has been widely used as a model for the binding of natural ligands to proteins. Experimental results have shown that each molecule of albumin binds at least six molecules of phenol red. The presence of fatty acids such as decanoate, palmitate, stearate, and oleate... 

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. 

Fig. 9 Structure of Phenol Red-modified porphyrin that enhances the binding affinity with proteins.

There have been many reports in which the immobilization method was covalent binding. In fact, many pH indicators used in above reports own at least one active amino or carboxyl group so that they can be covalently bound relatively easily to a solid substrate [165,166], Kostov et al. had discussed the immobilizing process of Congo red, neutral red and phenol... 

Serum albumin circulates in the blood stream transporting essential nutrients such as fatty acids to peripheral tissue. Transported molecules, called ligands, often have a special affinity for selected binding sites on proteins and nucleic acids. In this experiment, the dynamics of ligand-protein interactions will be explored with the binding of the dye phenol red to bovine serum albumin. The technique of gel filtration will be used to separate the dye-protein complex. Data will be analyzed in order to construct binding curves. 

In this experiment, you will evaluate the binding of the dye phenol red to bovine serum albumin. Increasing amounts of dye will be added to fixed amounts of protein in buffered solutions. The reaction mixture will be subjected to gel filtration to separate the dye-protein complex from free, unbound dye. Two types of binding studies can be completed ... 

Alcohol oxidation requires release of a proton, which formally comes from the alcohol. In other dehydrogenases such as lactate dehydrogenase, proton release occurs simultaneously with hydride transfer. In liver ADH proton release can be demonstrated, by reaction of the proton with an indicator such as thymol blue or phenol red in stopped-flow spectrophotometry, to be faster than hydride transfer, 270 vs. 150 s and unaffected by use of deuterated substrate, so it occurs before hydride transfer. Binding of the NAD+ nicotinamide ring is accompanied by a conformational change of ADH bringing the catalytic zinc about 0.1 nm closer to the... 

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. 

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