Determination of Protein Using Biuret Reagent - Assignment Example

The methods used are all-optical methods. This means that the level of protein concentration depends on optical characteristics (turbidity or absorption). These optical properties are measured using a tool called a colorimeter or spectrophotometer. Other useful methods are volumetric, gravimetric, among others (Slater, 1986, 12). After the reaction between the reagents and the protein substrate, the peptide bond turns purple or violet for positive results. We cannot conclude that there is the presence of protein by candid colorimetric methods.

This is because proteins are colorless. However, absolute estimations can be made to analyze the protein in the solution (Slater, 1986, 12). A stable color is achieved. It is always advisable to take all reading within the first 10mins for the purpose of clarity. In many experiments, biuret can be scaled using smaller sized cuvette, which consumes less protein. Proteins with abnormally low or a high percentage of amino acids and aromatic side groups always give low or high readings, respectively (Robinson, and Hogden, 1940, 32) In the case of bovine serum, albumin will obviously develop a linear relationship between the amount of protein and absorbance over a range of 0.

5 to 20mg protein. However, the analysis show assay below 0.5 mg is not reliable. In other cases, the upper limit may increase depending on the reagents. A straight-line graph is achieved after plotting the relationship between protein concentration and absorbance. This means that there is a tremendous increase in absorbance with an increase in protein concentration (Layne, 1957, 23). The reagent is made up of potassium hydroxide. It also has traces of hydrated copper (II) sulfate and potassium sodium titrate.

The blue color appears as a result of the presence of proteins, pink and blue may also arise in the presence of short-chained polypeptides. A biuret reagent is not required in all protein tests.