Estimation of Tyrosine by Folin-Ciocalteau Method
Estimation of Tyrosine by Folin-Ciocalteau Method:
The Folin–Ciocalteu reagent (FCR) or Folin’s phenol reagent or Folin–Denis reagent, also called the Gallic Acid Equivalence method (GAE), is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric in vitro assay of phenolic and polyphenolic antioxidants. It is named after Otto Folin, Vintilă Ciocâlteu, and Willey Glover Denis.More
The reagent does not only measure phenols, and will react with any reducing substance. It therefore measures the total reducing capacity of a sample, not just phenolic compounds. This reagent is part of the Lowry protein assay, and will also react with some nitrogen-containing compounds such as hydroxylamine and guanidine. The reagent has also been shown to be reactive towards thiols, many vitamins, the nucleotide base guanine, the trioses glyceraldehyde and dihydroxyacetone, and some inorganic ions. Copper complexation increases the reactivity of phenols towards this reagent.
Tyrosine because of presence of hydroxyl phenol group reduce the phospho molybdate present in the F/C reagent to form Blue color, which can be measured colorimetrically at 700 nm (or) by using red filter.
0.5N NaOH Solution: 20gms/100ml
Folin-Ciocalteau reagent: Reflux gently for 10 hours a mixture consisting of 100g sodium tungstate (Na2WoO4.2H2O), 25g sodium molybdate (Na2MoO4.2H2O), 700mL water, 50mL of 85% phosphoric acid, and 100mL of concentrated hydrochloric acid in a 1.5L flask. Add 150g lithium sulfate, 50mL water and a few drops of bromine water. Boil the mixture for 15min without condenser to remove excess bromine. Cool, dilute to 1L and filter. The reagent should have no greenish tint. (Determine the acid concentration of the reagent by titration with 1N NaOH to a phenolphthalein end-point).This is commercially available and has to be diluted with equal volume of water just before use.
Standard Tyrosine solution: 5mg/ml Bovine Serum Albumin (commercially available-used as Standard)
Take 0.2, 0.4, 0.6, 0.8 and 1ml into series if five test tubes add water to bring the volume of one ml in each test tube. Add 5ml of 0.5N NaOH solution and finally 1.5ml of dilute F/C reagent to each test tube mix well and measure the O.D. with a photoelectric colorimeter at 700nm (or) by using a red filter with in 2 to 10 minutes.
Prepare a Blank with 1 ml of distilled water instead of Tyrosine solution construct the standard curve with micrograms of tyrosine. Tyrosine concentration on the X-axis and OD on Y-axis using a standard curve determine the amount of tyrosine present in a given unknown solution.
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