Protein can be estimated by different method as by Lowry’s and also by estimating the total nitrogen content no method is 100% sensitive hydrogen the protein and the estimating the amino acids alone will give the extract give a moderately constant value and hence and here large followed protein content of enzymes extract is usually determine by this method.
The blue colour developed by the reduction of the phosphomolybdic phosphotingstic component in the folin- ciocatleau reagent by the amino acids tyrosine and tryptophan present in the protein plus the colour developed by the burette reaction and the protein with the alkaline tartarate are measure in the Lowry’s method.
Reagents required: 2% sodium carbonate in 0.1N sodium hydroxide (Reagent…show more content… A part from being bound as proteins, aminoacids also exist in the free forem in many tissues and are known as free aminoacids. They are mostly water soluble in nature, very often in plants during disease conditions. The free aminoacid composition exhibits a change and hence the measurement of the total free aminoacids gives the physiological and health status of the plants. Ninhydrin, a powerful oxidising agent, decarboxylates the α-aminoacids and yield an intensely coloured bluish purple product which is colorimetrically measured at…show more content… Hydrindantin + Ninhydrin+ Ammonia→ Purplre coloured product + water.
Ninhydrin: Dissolve 0.8g of Stannous chloride(SnCl2.2H2O) in 500ml of 0.2M Citrate buffer(pH 5.0). Add this solution to 20g Ninhydrin in 500ml of Methyl cellusolve (2methoxy ethanol). 0.2M Citrate buffer pH5.0 Diluent solvent: Mix equal volumes of water and n-propanol and use.
Extraction of aminoacids:
Weigh 500mg of the plant sample and grind it in a pestle and mortar with a small quantity of acid-washed sand. To this homogenate, add 5 to 10ml of 80% ethanol, filter or centrifuge. Save the filterate or supernatant. Repeat the extraction twice with the residue and pool all the supernatants. Reduce the volume if needed by evaporation and use the extract for the quantitative estimation of total free aminoacids. If the tissue is tough, use boiling 80% ethanol for