Quantitative Protein Determination

Introduction

Biuret solution can be used with a spectrophotometer or colorimeter to quantitatively determine protein concentration.

Concepts

• Proteins
• Peptide bonds
• Beer’s Law
• Spectrophotometric analysis

Materials

Safety Precautions

Biuret solution contains copper sulfate in a sodium hydroxide solution. It is corrosive to all body tissue, especially eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Solutions should be neutralized using dilute hydrochloric acid solution and flushed down the drain with excess water according to Flinn Suggested Disposal Method #10.

Prelab Preparation

Procedure

1. Number seven spectrophotometer test tubes 1 to 7.
2. Add 5 mL distilled water to test tube 1. This is the “blank.”
3. Add 5 mL of solution 2 to test tube 2; 5 mL of solution 3 to test tube 3, etc.
4. Add 2.5 mL of biuret solution to each of the test tubes. Mix by rotating the tubes between the palms of your hands.
5. Wait 30 minutes for any color to fully develop. The fully developed color is stable for at least an hour. During this time, standardize the spectrophotometer or colorimeter using tube 1 as the blank (100% transmittance). Set a wavelength of 540 nm on the spectrophotometer, or the closest wavelength available on the colorimeter (565 nm).
6. Determine the % transmittance (%T) and absorbance (A) for tubes 2–5. Plot absorbance of the solution versus concentration of albumin in the solution. This will provide a standard Beer’s Law-type plot for the albumin solutions.
7. Using the Beer’s Law plot obtained in Step 6, determine the theoretical concentrations of the “unknowns” (tubes 6 and 7). How do the theoretical predictions compare to the actual predictions?

Teacher Tips

• Biuret solution can also be used as a simple qualitative test for proteins. A pinkish or purplish violet color indicates the presence of proteins with at least two peptide linkages. Proteoses and peptones give a pink color; gelatin turns a bluish color.

Discussion

Proteins are chains of amino acids connected by peptide bonds. Proteins and other smaller chains of amino acids are sometimes referred to as polypeptides. When biuret solution is added to a solution containing polypeptides, the copper ions in the solution react with the peptide bonds to produce a pinkish or purplish color.

The biuret molecule (prepared from urea) is not a protein, but it contains two peptide bonds. Therefore, it gives a positive test when reacted with biuret solution.

It is important to note that biuret solution does not contain the biuret molecule; it merely contains copper sulfate in a sodium hydroxide solution, and is the test solution for multiple peptide-containing molecules, such as biuret and proteins.

References

Abramoff, P.; Thomson, R. G. Laboratory Outlines in Biology—V; W. H. Freeman: New York, 1991; pp 124–127.

Morholt, E.; Brandwein, P. F. A Sourcebook for the Biological Sciences, 3rd ed.; Harcourt Brace Jovanovich: Fort Worth, TX, 1986; p 179.