In this article we will discuss about the tests for detection of proteins and amino acids in plants with the help of experiments.
Proteins and other elated nitrogenous compounds are the principle constituents of protoplasm and are directly involved in all the physiological processes taking place in the living cells. Proteins of all kinds are synthesized from amino acids, which are joined one after another by peptide linkage.
Proteins are colloidal in nature and do not readily diffuse through the plasma membrane. Almost all proteins are irreversibly coagulated by heat. Proteins are insoluble in neutral salts such as NaCl, MgSO4 etc. in which they precipitate without change in composition, but go into solution on further dilution of the salt.
Some are soluble in alkali while basic proteins are soluble in acid. The amino acids are amphoteric compounds have a basic amino moiety and an acidic carboxyl moiety and they are highly reactive. The amino acids are mostly soluble in water.
Requirements for Detecting Proteins and Amino Acids in Plants:
(a) Preparation of Sample Solution:
Protein solution is prepared in water with phosphate buffer. From plant sources soluble proteins are easily extracted by distilled water, while insoluble ones are dissolved by alkaline treatment.
Amino acids are, however, soluble in water. The common plant materials used as proteins and free amino acids sources are wheat, flour, germinating seeds and crushed seeds etc. The extract is finally filtered for detection of proteins or amino acids.
(b) Reagents:
1. NaOH solution — 5 gm. in 100 ml dist. water
2. CuSO4 solution — 1 gm. in 100 ml dist. water
3. Concentrated HNO3
4. Ninhydrin solution — 5 gm. in 100 ml dist. water
5. Millon’s reagent — 5 gm. of Hg in 95 ml conc. HNO3, then dilute to 200 ml by addition of dist. water after dissolution of Hg
6. Lead acetate solution — 5 gm. in 100 ml dist. water
7. HgCl2 solution — 5 gm. in 100 ml dist. water
8. NaOH solution — (40%)
9. Glyoxylic acid
Tests for Detection of Proteins and Amino Acids in Plants:
The following tests can be performed for detection after preparation of sample solution from salt or plant materials:
(A) Tests for Amino Acids:
Experiment:
1. Ninhydrin Test:
To a small amount of extract a few drops of 5% Ninhydrin solution is added. The solution is heated in a water bath for 10 mins.
2. Test for Tyrosine :
A few drops of Millon’s reagent is added to the sample solution and then heated.
3. Test for tryptophan :
A few drops of glyoxylic acid and H2SO4 are added to the sample solution.
4. Test for Cysteine :
About 5 ml of sample solution is boiled with a little of 40% NaOH and a few drops of % lead acetate solution.
Observation:
The solution turns purple or bluish.
The solution turns dark red.
A reddish-violet ring at the junction of the two liquids is developed.
There is black precipitate of lead sulphate.
Inference:
The sample contain amino acids
Presence of Tyrosine.
Presence of Tryptophan.
Presence of Cysteine.
(B) Tests for Proteins:
Experiment:
1. Biuret Test:
To about 3 ml of sample solution, 1 ml of 4% NaOH soln. and a few drops of 1% CuSO4 solution are added.
2. Xanthoprotein Test:
To about 3 ml of sample solution, 1 ml of conc. H2SO4 is added.
3. Millon’s Test:
To about 3 ml of the protein solution 4-5 ml of Millon’s reagent is added.
4. Adamkicw’s Test:
To about 3 ml of sample solution, a few drops of acetic acid are added. Then conc. H2SO4 is added slowly to the sample.
5. Precipitation Test:
The protein can be precipitated from the sample solution by the addition of:
(a) Absolute alcohol
(b) 5% CuSO4 soln.
(c) 5% Pb-acetate soln.
(d) 5% HgCI2 soln.
(e) 5% (NH4)2SO4 soln.
6. Test for Sulphur Containing Protein:
About 5 ml of protein solution is boiled after addition of a few mis of 40% NaOH soln., and a drop or two of 10% lead acetate solution.
Observation:
A violet or pink colour is produced.
A white precipitate is formed. On boiling, the ppt. turns yellow. On addition of NH4OH soln. the ppt. becomes orange.
A white ppt. is formed. On warming the ppt. turns brick red, or it may be dissolved, giving a red solution.
A violet colour develops at the junction of the two liquids.
White colloidal precipitate is formed.
The solution turns black or brownish due to the formation of PbS by the action of strong alkali on sulphur of the protein.
Inference:
This is a general test for all proteins containing the CONH group.
Presence of protein containing Tryptophan or Tyrosine.
Presence of protein.
Presence of protein containing Tryptophan.
Presence of all kinds of protein.
This is a positive test for the sulphur containing amino acids and proteins.