Determination of Protein by Ninhydrin Method part 1

Aim of Experiment To indicate protein which can be determined by visual eyes or by spectrophotometer that shows a violet color of the sample when they’re reacted.

Theory Amino acids are known as the building blocks of all proteins. There are 20 different amino acids commonly found in proteins. Ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color that is detected in this method. Ninhydrin will react with a free alpha-amino group, NH 2 -C-COOH. This group is present in all amino acids, proteins or peptides. Whereas, the decarboxylation reaction will proceed for a free amino acid , it will not happen for peptides and proteins. The primary amino groups react with Ninhydrin to form the purple color dye now called Ruhemann's purple (RP) was discovered by Siegfried Ruhemann in 1910. amino acids like proline, the guanidine group of arginine, the amide groups of asparagine, the indole ring of tryptophan, the sulfhydryl group of cysteine, amino groups of cytosine and guanine, and cyanide ions also react with Ninhydrin to form various chromophores that can be analyzed. In the quantitative estimation of amino acid using Ninhydrin reagent, the absorbance of the Ruhemann's purple formed by the reaction at 570 nm is measured. For amino acids, the absorption happens at 440 nm.

Tools & Apparatus Pipettes 100 (ml) & 50 (ml) Beakers test tubes (5 -10 ml) quartz cuvette Spectrophotometer Hotplate Glass rod Watch glass Spatula Sensitive Balance Timer or stop watch

Sample preparation Glycin solution Measure (0. 250, 0. 500, 0. 750) g of Glycin on the sensitive balance separately. Put it into the beaker. Add 10 ml distilled water to the beaker. Mix them well by shaking or a rod. until you get a complete solution of Glycin Solution. Proline Solution Measure (0. 250, 0. 500, 0. 750)g of Proline Powder on the sensitive balance Separately. Put it into the beaker. Add 10 ml distilled water to the beaker. Stir them well by shaking or a rod. until you get a complete solution of Proline Solution Reagent Preparation Ninhydrin solution • Measure 0. 025 g of ninhydrin reagent on sensitive tare. • Put it into the beaker. • Add 5 ml of acetone. • Mix them well till you get yellow solution which is Ninhydrin Solution

Procedure Pipette out the samples in the respective labelled test tubes about 1 ml of solution. Add 1 ml of distilled water to the test tube labelled Blank. Now add 2 -3 drops of Ninhydrin reagent to all the test tubes including the test tubes labelled 'blank' and 'unknown'. Mix the contents of the tubes by vortexing/shaking the tubes. Place all the test tubes in boiling water bath for 5 -10 minutes Cool the test tubes in cold water up to 3 minutes. Now Samples are ready to use in spectrophotometer.