Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Quantitative Protein Determination
Protein Determination Using the Biuret Reaction

8.18.3.1. Micromethod [180].

Reagents.

A. 0.21% CuSO₄∙5H₂O solution in 30% NaOH. To prevent copper hydroxide precipitation, this solution is prepared as follows: 21 ml of a 1% (w/v) aqueous solution of CuSO₄∙5H₂O is added to 75 ml of 40% NaOH, and the mixture is then diluted with Water to 100 ml.

Б. 30%-ный NaOH.

Both reagents can be stored in Pyrex glassware for up to 6 months.

Procedure. To 2 ml of protein solution (0.02–0.53 mg/ml), add 1 ml of Reagent A. Mix thoroughly and let stand at room Temperature for 5 min, then measure the absorbance at 310 nm against a blank. Prepare the following mixtures:

X₁ — 2 ml of protein solution + 1 ml of Reagent A;

X₂ — 2 ml of distilled water + 1 ml of Reagent A;

Y₁ — 2 ml of protein solution + 1 ml of Reagent B;

Y₂ — 2 ml of distilled water + 1 ml of Reagent B.

Protein absorbance equals (X₁—X₂) — (Y₁—Y₂); use a calibration curve.

Note. This method is a Modification of the one previously described [417]; the procedure can be used in the presence of DNA. DNA concentrations up to 0.7 mg/ml in the reaction mixture do not interfere with the assay, i.e., a DNA-to-protein ratio of ≥10:1 is permissible.

Color development takes less than 5 min and remains stable for 2.5 h (for the seven Proteins studied); in the case of Lysozyme, an 11% decrease in color intensity was observed over this time. The sensitivity is 1/16 that of the Lowry Method, but a significant advantage is that the absorbance-to-protein concentration relationship is linear, and the Influence of the specific protein nature is less pronounced. The reaction is unaffected by the presence of NaCl (1.5 M), sodium acetate (0.1 M), sodium formate (0.75 M), perchloric acid (0.67 M), or Na₂H phosphate (0.01 M) in the solution. At urea concentrations >2.0 mol/l, a small correction for urea absorbance is required.

An interesting method has been proposed in which the protein is treated first with the biuret reagent and then with the Folin reagent. Good reproducibility was achieved (±2% for a protein concentration of 50–600 µg/ml). In Hill coordinates [284], the absorbance versus protein concentration curve transforms into a straight line.

8.18.3.2. Biuret method for thiol-containing samples [291].

Reagent. Dissolve 3.9 g of CuSO₄∙5H₂O and 6.7 g of Na₂-EDTA in 700 ml of distilled water. Add 200 ml of 20% NaOH with constant stirring to a final volume of 900 ml.

Procedure. A solution containing 50–1000 µg of protein is diluted to a volume of 0.1 ml in a test tube, 1.0 ml of biuret reagent is added, the mixture is incubated for 5 min at 60 °C, and the absorbance is measured at 545 nm.

Note. The method has moderate sensitivity (~50 µg), but is simple and yields a linear relationship between absorbance and concentration up to 1000 µg of protein. The color is stable for at least 1 h. The addition of iodoacetamide (12.5 µg) is necessary to eliminate the Interference of large amounts of dithiothreitol. The EDTA reagent [66] surpasses tartrate [129] in chelating capacity, completely eliminating The Effect of sulfhydryl compounds.

The copper-diethyldithiocarbamate complex in the presence of deoxycholate yielded a linear plot for BSA at protein concentrations up to 500 µg/ml [185].

8.18.3.3. Biuret method applied to detergent-containing samples [393].

Reagent. 1.5 g CuSO₄∙5H₂O + 4.9 g Na₂-tartrate dihydrate + 7.5 g NaOH in 1 L of water. The reagent is stable for 4 months when stored in a plastic container at room temperature.

Procedure. To 2.5 ml of biuret reagent, add 0.5 ml of a solution containing 0.2–0.4 mg of protein and 2% detergent. Mix and let stand at room temperature for 30 min. Measure the absorbance at 540 nm against 0.5 ml of a 2% detergent solution.

The modified reagent prevents solution turbidity in the presence of detergents used for Protein Extraction. A linear dependence of absorbance on protein content is maintained for BSA in the range of 0.4–8 mg/ml.

8.18.3.4. Biuret method applied to samples obtained by tissue extraction [24].

Procedure. The reagent described in Section 8.18.3.3 is used [393].

The aqueous tissue extract is washed with 3 ml of an acetone – petroleum ether mixture (1:1) at 35–60 °C with stirring on a vortex mixer for 30 s. Centrifuge and decant. Add 0.1 ml of 10% deoxycholate (pH 8.0) and 2.9 ml of reagent to the residue. Close the tube with Parafilm and mix the solutions by gently inverting the tube. Sonicate twice for 15 s at 20 kHz using a Branson Sonifier ultrasonic source. For maximum color development, place the tube in a boiling water bath for 30 s. Cool to room temperature and measure the absorbance at 540 nm against a reagent blank.