PAG 9.1

"Qualitative Testing for Proteins" (Biuret Test)

Positive test for Proteins: PurpleNegative test for Proteins: Remains blue

- Add a few drops of Sodium Hydroxide Solution (To make the solution alkaline)- Add Copper Sulfate + Potassium Hydroxide Solution (Biuret)

Extension Questions:1) What is the chemical composition of Biuert Reagent and which of these chemicals are hazardous? - Made up of Potassium Hydroxide and Copper Sulfate, which contains Copper ions. - Potassium Hydroxide is especially dangerous - this makes the solution alkali without the help of Sodium Hydroxide.2) Why does the procedure include making observations with the Biuret as a layer on top of the sample before mixing?- Protein suspension is cloudy - more difficult to see a colour change if the two are mixed straight away. - If samples only had small amount of protein (Short polypeptide chains / peptide bonds) then the colour change will not be as strong, making it more difficult to see (it would only go a weak pink colour instead of purple). - Eg. If mixed straight away, the purple could become mistaken for pink due to the cloudiness of the suspension. 3) What is the biochemical explanation for the positive results with the biuret test?- Key component to colour change, is peptide bonds. - Lone electrons from 4 Nitrogen atoms in the peptide bond, coordinate a Copper ion to form the chelate complex. - Chelate complex absorbs light at 540nm - making it appear violet. - The greater the concentration of peptide bonds, the greater the colour intensity.

More Questions from the Books:1) Suggest a suitable liquid that may be used in the control tube? Distilled Water - No possibility of peptide bonds - Proves that it's the protein's peptide bonds that cause the colour change. 2) Explain why a protein is refferred to as a suspension, rather than a solution?