Biuret Assay

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Introduction

The term "biuret" describes the family of organic compounds with the functional group (HNCO)2N-R

The term "biuret" describes the family of organic compounds with the functional group (HNCO)2N-R

The Biuret Assay, also known as the Piotrowski Test, is a biochemical assay that allows one to accurately quantify protein concentration within the range of 5-150 mg/mL. The protein sample, irrespective of its composition, is measured through absorbance spectroscopy at 540 nm in conjunction with a known protein concentration sample. 

The advantages to this method is that the turn around time is realtively short with few interfering substances. The disadvantages include low sensitivity, and that ammonium sulfate can often interfere with color development or generate colored complexes within the sample. This can be minimized by analyzing protein precipitates prior to performing the test. 

Principle

Polypeptide structure compared to Urea and Biuret. via ThermoFisher [2]

Polypeptide structure compared to Urea and Biuret. via ThermoFisher [2]

In alkaline solutions containing sodium potassium tartrate, cupric ions complex with the peptide bonds of proteins forming a light-blue to purple colored complex. This is known as the biuret reaction because biuret is a byproduct of excess urea and heat that forms with copper ions producing a similar colored complex in solution.  Because the structure of polypeptides is similar to both urea and biuret, it is able to complex with copper under proper conditions creating the same colorimetric change.

Because the number of peptide bonds per unit weight is the same for all proteins, and the reaction only occurs with peptide bonds and not amino acid side chains, the color intensity of the sample is directly proportional to protein concentration.  

Model structure of Bovine Serum Albumin via Chinnathambi et al. [3]

Model structure of Bovine Serum Albumin via Chinnathambi et al. [3]

A standard curve using a similar protein is created for comparison during testing due to the unknown composition of the unknown sample. The best protein to use is a purified preparation of the specific protein, however, due to rare availability, a relative standard is often used to provide a similar color yield.  An aqueous solution of bovine serum albumin (BSA) is commonly used as a standard in testing because of its stability in testing, low cost, and is readily available as a byproduct from bovine blood. 

Equipment and Materials

Equipment

  • 1 Liter Volumetric Flask
  • 1 Liter Beaker
  • Disposable or Reusable Cuvettes.
  • Micropipettors
  • Suitable tubes to mix and hold 3.0 mL samples
  • Spectrophotometer
  • Stir Rod and/or Magnetic Stir Plate
  • Vortex

Reagents

  • Copper (II) sulphate pentahydrate
  • Distilled or Deionized Water
  • Potassium sodium tartrate tetrahydrate
  • Potassium Iodide
  • Sodium Hydroxide 
  • Stock Solution of Bovine Serum Albumin (10 mg/mL) or a standard protein solution of known concentration.

Preparation of the Biuret Reagent

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  1. Weigh and dissolve 1.5 g of copper (II) sulphate pentahydrate and 6.0 g of sodium potassium tartrate in 500 mL of water.
  2. Add 300 mL of 10% (w/v) sodium hydroxide.
  3. Fill remainder of volume to 1 Liter with water.
  4. Add 1.0 g of potassium iodide to inhibit the reduction of copper. (optional)
  5. Store reagent in a plastic container away from light.

Discard if black or red precipitate is observed in solution

Method

  1. Turn on the spectrophotometer and associated software and allow to warm up prior to testing. (Read instruction manual for designated equipment for proper techniques when setting up or troubleshooting)
  2. Prepare serial dilutions of the unknown and standard samples ranging in concentration from 0.5 to 10 mg/mL so that the final volume for the assay is 0.5 mL. 
  3. Add 2.5 mL of biuret reagent to each sample for a total volume of 3.0 mL.
  4. Vortex samples briefly and allow both unknown and standard samples to incubate at room temperature for 30 minutes. 
  5. Transfer samples to cuvettes and measure absorbance at 540 nm starting with the lowest dilutions. Record values either directly onto a spreadsheet or in a lab notebook.

Analysis

Create a standard curve by plotting the concentration and absorbance values. Create a linear regression to determine the equation that best fits the values found similar to the image below:

Note: These values depict a low concentration of protein. Due to sensitivity of the test, only perform with samples in which original protein concentrations range from 5-150 mg/mL.

Note: These values depict a low concentration of protein. Due to sensitivity of the test, only perform with samples in which original protein concentrations range from 5-150 mg/mL.

After construction of a standard curve, use the value associated with the linear regression and backtrack to find the concentration of the sample. 


References

  1. "Biuret Protein Assay." Jenway. Bibby Scientific. http://www.jenway.com/adminimages/P09_002A_Biuret_Protein_Assay.pdf
  2. “Chemistry of Protein Assays | Thermo Fisher Scientific - US.” Thermo Fisher Scientific, Thermo Fisher Scientific, www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chemistry-protein-assays.html.
  3. Chinnathambi, Shanmugavel & Karthikeyan, Subramani & Velmurugan, Devadasan & Hanagata, Nobutaka & Aruna, Prakasarao & Ganesan, Singaravelu. (2015). Effect of Moderate UVC Irradiation on Bovine Serum Albumin and Complex with Antimetabolite 5-Fluorouracil: Fluorescence Spectroscopic and Molecular Modelling Studies. International Journal of Spectroscopy. 2015. 1-12. 10.1155/2015/315764. 
  4. "HiPer Protein Estimation Teaching Kit (Quantitative)." Himedia Labs. http://himedialabs.com/td/htbc005.pdf
  5. Martina, Vrsanska & Vojtech, Kumbar. (2015). A Comparison of Biuret, Lowry, and Bradford Methods for Measuring the Egg's Proteins. MendelNet. https://mnet.mendelu.cz/mendelnet2015/articles/62_vrsanska_1167.pdf
  6. “Which Protein Assay Is Best for You?” Azure Biosystems, 10 Aug. 2017, www.azurebiosystems.com/protein-assay-best/.