Method 2

This method (commonly referred to as the Lowry assay) is based on the reduction by protein of the phosphomolybdotungstic mixed acid chromogen in the phosphomolybdotungstic reagent, which results in an absorbance maximum at 750 nm. The phosphomolybdotungstic reagent reacts primarily with tyrosine residues in the protein. Colour development reaches a maximum in 20 min to 30 min at room temperature, after which there is a gradual loss of colour. Because the method is sensitive to interfering substances, a procedure for precipitation of the protein from the test sample may be used. Most interfering substances cause a lower colour yield; however, some detergents cause a slight increase in colour. A high salt concentration may cause a precipitate to form. Because different protein species may give different colour response intensities, the reference substance and test protein must be the same. Where separation of interfering substances from the protein in the test sample is necessary, proceed as directed below for interfering substances prior to preparation of the test solution. The effect of interfering substances may be minimised by dilution, provided the concentration of the test protein remains sufficient for accurate measurement.

Use distilled water R to prepare all buffers and reagents used for this method.

Test solution. Dissolve a suitable quantity of the substance to be examined in the prescribed buffer to obtain a solution having a concentration within the range of the standard curve. A suitable buffer will produce a solution of pH 10.0 to 10.5.

Reference solutions. Dissolve the reference substance for the protein to be determined in the prescribed buffer. Dilute portions of this solution with the same buffer to obtain not fewer than five reference solutions having protein concentrations evenly spaced over a suitable range situated between 5 Mg/ml and l0o Mg/ml.

Blank. Use the buffer used to prepare the test solution and the reference solutions.

Copper sulphate reagent. Dissolve 100 mg of copper sulphate R and 0.2 g of sodium tartrate R in distilled water R and dilute to 50 ml with the same solvent. Dissolve 10 g of anhydrous sodium carbonate R in distilled water R and dilute to 50 ml with the same solvent. Slowly pour the sodium carbonate solution into the copper sulphate solution with mixing. Use within 24 h.

Alkaline copper reagent. Mix 1 volume of copper sulphate reagent, 2 volumes of a 50 g/l solution of sodium dodecyl sulphate R and 1 volume of a 32 g/l solution of sodium hydroxide R. Store at room temperature and use within 2 weeks.

Diluted phosphomolybdotungstic reagent. Mix 5 ml of phosphomolybdotungstic reagent R with 55 ml of distilled water R. Store in an amber bottle, at room temperature.

Procedure. To 1.0 ml of each reference solution, of the test solution and of the blank, add 1.0 ml of alkaline copper reagent and mix. Allow to stand for 10 min. Add 0.5 ml of the diluted phosphomolybdotungstic reagent, mix and allow to stand at room temperature for 30 min. Determine the absorbances (2.2.25) of the solutions at 750 nm, using the solution from the blank as compensation liquid.

Calculations. The relationship of absorbance to protein concentration is non-linear; however, if the range of concentrations used to prepare the standard curve is sufficiently small, the latter will approach linearity. Plot the absorbances of the reference solutions against the protein concentrations and use linear regression to establish the standard curve. From the standard curve and the absorbance of the test solution, determine the concentration of protein in the test solution.

Interfering substances. In the following procedure, deoxycholate-trichloroacetic acid is added to a test sample to remove interfering substances by precipitation of proteins before determination; this technique can also be used to concentrate proteins from a dilute solution.

Add 0.1 ml of a 1.5 g/l solution of sodium deoxycholate R to 1 ml of a solution of the substance to be examined. Mix using a vortex mixer and allow to stand at room temperature for 10 min. Add 0.1 ml of a 720 g/l solution of trichloroacetic acid R and mix using a vortex mixer. Centrifuge at 3000 g for 30 min, decant the liquid and remove any residual liquid with a pipette. Redissolve the protein pellet in 1 ml of alkaline copper reagent.

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