Determination of protein content → biuret method

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Determination of protein content → biuret method

Purpose requirement

Learn the principles and methods of protein determination by biuret method.

Experimental principle

Compounds having two or more peptide bonds all have a biuret reaction. Biuret combines with copper ions in an alkaline solution to form a complex magenta complex. The peptide bonds of proteins and peptides are similar to those of biuret, and can also form a purple-red coordination compound with Cu2+, and its maximum light absorption is at 540 nm. Its color depth is proportional to the protein concentration, and the protein urea method is commonly used for rapid determination of proteins.

Reagents and equipment

1), reagent

Biuret reagent: 1.5 g of copper sulfate (CuSO4·5H2O) and 6.0 g of sodium potassium tartrate (NaKC4H4O6·4H2O) were dissolved in 500 mL of distilled water, 300 mL of 10% NaOH solution was added with stirring, and diluted to 1000 mL with water. This reagent can be stored for a long time and used.

2), standard and protein solution to be tested

(1) Standard protein solution. 10 mg/mL crystalline bovine serum albumin solution or the same concentration of casein solution (prepared with 0.05 mol/L sodium hydroxide solution). As a standard protein, the protein content is determined in advance by a micro-Krebs method, and the standard solution is prepared by weighing according to its purity.

(2), the protein solution to be tested. Human serum (diluted 10 times). Test other protein samples should be diluted to the appropriate multiples to make them within the standard curve test range.

3), equipment

Tube 1.5 × 15 cm (× 16), test tube rack, pipette 1 mL (×3); 5 mL (×1); constant temperature water bath; spectrophotometer.

Precautions

(1), colorimetric determination within 30 minutes after color development. The time from color development to colorimetric of each tube should be as uniform as possible.

(2) When a large amount of fatty substances are present at the same time, a turbid reaction mixture may be generated. At this time, the solution may be clarified by ethanol or petroleum ether, and then centrifuged, and the supernatant is taken for measurement.