Protein estimation by Lowry’s method

Protein can be estimated by different methods as described by Lowry and also by estimating the total nitrogen content. No method is 100% sensitive. Hydrolyzing the protein and estimating the amino acids alone will give the exact quantification. The method developed by Lowry et al¹ is sensitive enough to give a moderately constant value and hence largely followed. Protein content of enzyme extracts is usually determined by this method.

Principle

The blue color developed by the reduction of the phosphomolybdic-phosphotungstic components in the Folin-Ciocalteau reagent by the amino acids tyrosine and tryptophan present in the protein plus the color developed by the biuret reaction of the protein with the alkaline cupric tartrate are measured in the Lowry’s method.

Materials

» 2% Sodium Carbonate in 0.1N Sodium Hydroxide (Reagent A)
» 0.5% Copper Sulphate (SuSO₄.5H₂O) in 1% potassium sodium tartrate (Reagent B)
» Alkaline Copper Solution: Mix 50mL of A and 1mL of B prior to use (Reagent  C)
» Folin-Ciocalteau Reagent (reagent D) – 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).
» Protein Solution (Stock Standard)
Weigh accurately 50mg of bovine serum albumin (Fraction V) and dissolve in distilled water and make up to 50mL in a standard flask.
» Working Standard
Dilute 10mL of the stock solution to 50mL with distilled water in a standard flask. One mL of this solution contains 200mg protein.

Procedure

Extraction of Protein from Sample
Extraction is usually carried out with buffers used for the enzyme assay. Weigh 500mg of the sample and grind well with a pestle and mortar in 5-10mL of the buffer. Centrifuge and use the supernatant for protein estimation.

Estimation of Protein

1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard into a series of test tube.
2. Pipette out 0.1mL and 0.2mL of the sample extract in two other test tubes.
3. Make up the volume to 1mL in all the test tubes. A tube with one mL of water serves as the blank.
4. Add 5mL of reagent C to each tube including the blank. Mix well and allow to stand for 10min.
5. Then add 0.5mL of reagent D, mix well and incubate at room temp. in the dark for 30min. Blue color is developed.
6. Take the reading at 660nm.
7. Draw a standard graph and calculate the amount of protein in the sample.

Calculation

Express the amount of protein mg/g or 100g sample.

Notes

1. For complete enzyme extraction, sometimes the chemical like ethylenediamine tetraacetic acid (EDTA), magnesium salts and mercaptoeethanol are included. This method of protein estimation should not be followed if the extractant K⁺, Mg⁺⁺, Tris, EDTA and thiol (mercaptoethanol) compounds as they interfere with this procedure.
   When this chemicals are present in the extract, precipitate the protein by adding 10% TCA, centrifuge and dissolve the precipitate in 2N NaOH and proceed for protein estimation.
2. If the protein concentration of the sample is high (above 500mg/mL) measure the color intensity at 550nm.
3. Rapid mixing as the Folin reagent is added is important for reproducibility.
4. A set of standards is needed with each group of estimation preferably in duplicate. Duplicate or triplicate unknown are also recommended.
5. Folin-Ciocalteau reagent can be purchased commercially. Store refrigerated in amber bottles. A good quality reagent is straw yellow in color.
6. If protein estimation is desired in a sample with high phenolic or pigment content, extract should be prepared with a reducing agent preferably cysteine and NaCl. Precipitate the protein with TCS, separate the protein and dissolve in 2N NaOH and proceed.

References

1. Lowry, O H, Rosebrough, N J, Farr, A L and Randall, R J (1951) J Biol Chem 193 265.
2. Mattoo, R L (1970) India J Biochem 7 82.