Folin reagent

A method that has been the standard of choice for many years is the Lowry procedure. This method uses Cn ions along with Folin-Ciocalteau reagent, a combination of phosphomolybdic and phosphotnngstic acid complexes that react with Cn. Cn is generated from Cn by readily oxidizable protein components, such as cysteine or the phenols and indoles of tyrosine and tryptophan. Although the precise chemistry of the Lowry method remains uncertain, the Cn reaction with the Folin reagent gives intensely colored products measurable spectrophotometrically. 

The older methods for the measurement of protein in natural waters usually depended upon the presence of aromatic amino acids in the protein, and calculated total protein on the basis of an average tyrosine, tryptophan, or phenylalanine content. A method representative of this type was the Folin reagent method published by Debeika et al. [281]. While these methods were useful in fresh water and in some coastal regions, they were not sensitive enough for the lower concentrations to be found in oceanic waters. 

Lowry O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin reagent. 

Dinitrofluoro benzene Isatin-zinc acetate Folin reagent Ninhydrin... 

Dilutions of a 400-ppm chlorogenic acid solution were used to obtain a calibration curve for the FC assay. One milliliter of 0.25 N Folin reagent was added to 1 mL of the diluted chlorogenic acid solution. After mixing, the solution was allowed to stand for 3 min. One milliliter of sodium carbonate (1 N) was then added, mixed, and allowed to stand for another 7 min. Subsequently, 7 mL of deionized water was added, mixed, and allowed to stand for 2 h. The absorbance of the mixture was read on a spectrophotometer (HP 8452A Diode Array) at a wavelength of 726 nm and plotted as a function of the chlorogenic acid concentration. Samples of crude and fractionated plant extracts were analyzed in a similar manner. 

Contrasting with the aforementioned assessments, the SC can be studied after staining the harvested SACD. Many dyes are available and their choice depends on the goal of the study. As an example, a mixture of rhodamine B and methylene blue conveniently decorates the corneocytes for microscopic examination. Another quantitative assay is based on the Lowry s reaction of proteins with an alkaline copper tartrate solution and Folin reagent followed by spectrophotometric measurement of the reaction product with maximum absorbance at 750 nm 59... 

Figure 2. Leakage of a, folin-reagent and b, phenol-HjS04 positive substances from S. cerevisiae cells during incubation with polygodial. o-o. None c-c, 1 /xg/ml of polygodial 10...

With reagents prepared in advance, the Lowry assay requires 1 h. A 400-pL sample is required, containing 2-100-pg protein (5-250 pg/mL). Nonlinear calibration curves are obtained, due to decomposition of the Folin reagent at alkaline pH following addition to the sample that results in incomplete reaction. Interferences include agents that acidify the solution, chelate copper, or cause reduction of cop-per(II). 

This is a modification of the Folin method, without the Folin reagent In this case the Cu formed does not mediate reduction of the Folin reagent but is chelated by bicinchoninic add. 

An analogous reaction occurs with 1,2-naphthoquinone-4-sulfonic acid Folin reagent) but, instead of a yellow color (cf. Formula 1.36), a red color develops ... 

The final colour in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phos-phomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue colour. The amino acid residues which are involved in the reaction are tryptophan and tyrosine as well as cysteine, cystine and histidine. The amount of colour produced varies slightly with different proteins. In this respect it is a less-reliable assay than the biuret method, but it is more reliable than the absorbance method since A280 may include contribution from other species, and also the absorption of a given residue is dependent on its environment within the protein. 

To perform an assay add x cm of sample (where X < 0.6) containing 5-100 pg of protein as required to (0.6 - x) cm of water. Then add 3 cm of the alkaline copper solution. The solutions must then be mixed well and allowed to stand for 10 min at room temperature. 3.0 cm of Folin reagent is then added and after 30 min the absorbance at 600 nm is determined. 

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