Laboratory Exercise 5: Tests for Proteins and Amino Acids Flashcards
Proteins, essential constituents of the living cells, are high molecular weight compounds which, upon hydrolysis, produce __.
amino acids
In a protein, the amino acids are linked by __.
peptide bonds
Common Tests for Proteins
- BIURET TEST
- HOPKINS-COLE TEST
- MILLON’S TEST
- XANTHOPROTEIC TEST
- REDUCED SULFUR TEST
- NITROPRUSSIDE TEST
- SAKAGUCHI TEST
Materials for Exercise 5
- Egg white
- Graduated cylinder (10 ml)
- Graduated cylinder (100 ml)
- Test tubes (35 pcs)
- Dropper (15 pcs)
- Beaker (250 ml, 2 pcs)
Test solutions for Exercise 5
A. 10% v/v egg white in water (10 mL egg white + 90 mL water)
B. 1% Tyrosine
C. 1% Tryptophan
D. 1% Phenylalanine
E. 1% Arginine
F. 1% Methionine
G. 1% Cysteine
Chemicals for Biuret Test
- 10% (w/v) NaOH
- 0.1% CuSO4 (copper(II) sulfate) solution
Chemicals for Hopkins-Cole Test
- Hopkins-Cole Reagent (Glyoxylic acid)
- Conc. Sulfuric acid (H2SO4)
Chemicals for Millon’s Test
- Millon’s Reagent
Chemicals for Xanthoproteic Test
- Concentrated nitric acid (HNO3)
- 10% (w/v) NaOH (same as in Biuret Test)
Chemicals for Reduced Sulfur Test
- 10% (w/v) NaOH (same as in Biuret Test)
- 1% Lead acetate
Chemicals for Nitroprusside Test
- 2% sodium nitroprusside
- 10% (w/v) NaOH (same as in Biuret Test)
Chemicals for Sakaguchi Test
- 40% (w/v) NaOH
- α-naphthol solution (1% w/v in ethanol)
- 10% sodium hypochlorite
a general test for proteins comparable to Molisch’s test for carbohydrates
biuret test
In the Biuret test, the compounds having two or more peptide bonds and copper salts in an alkaline solution create a __. In other words, the __ interacts with peptide bonds in an alkaline solution. These ions generate a __ when they react with the __ in the peptide link.
- purple color complex
- cupric ions (Cu2+)
- purple or violet-colored complex
- nitrogen
In the Biuret Test, __ is produced in an alkaline medium from copper sulfate from the Biuret reagent (__), the cupric ions of which interact with amide nitrogen, resulting in a violet or purple color complex.
- Cupric hydroxide
- 0.1% CuSO4 + NaOH
In the Biuret test, the chelate complex is purple in color due to its capacity to absorb light with a wavelength of __. As a result, the presence of proteins in the analyte is indicated by the development of a purple-colored complex. The amount of peptide bonds that are reacting, and hence the number of protein molecules present in the reaction system, determines the absorbance produced. In the Biuret test, short-chain peptides frequently produce a __ or __ color.
- 540 nm
- blue; pink
Hopkins-Cole Test test is a confirmatory test of the presence of __.
amino acid tryptophan (indole group)
The Hopkins-cole test is based on the principle that the layering of concentrated __ over a mixture of __, with Hopkins-Cole reagent results in the formation of __ or __ at the interface. The formation of the test color is due to the presence of __.
- sulfuric acid
- tryptophan-containing proteins
- purple or violet color ring
- indole group
does not respond to Hopkins-cole test due to absence of amino acid tryptophan.
Gelatin
The reagent used in Hopkins-Cole test contains __ (__) which condenses with __ in the presence of __ to form colored complexes (usually a violet interface is observed)
- glyoxylic acid; C2H2O3
- indole derivatives
- strong acids
This test is used to detect the presence of phenolic group-containing amino acids like tyrosine.
Millon’s Test
In Millon’s Test, the reaction takes place because of the __ and any phenolic compounds that are unsubstituted in the __ such as __, __, and __will give the positive reaction. The phenolic group containing amino acids reacts with __ solution (__) and forms a __ of the mercury-amino acid complex. After that, when __ solution is added and heat it until __ colored complex, __is produced.
- hydroxyphenyl group
- 3,5 positions
- phenol; tyrosine; thymol
- acidified mercuric sulfate
- mercury dissolved in nitric acid
- yellow precipitate
- sodium nitrate
- red
- mercury phenolate
The xanthoproteic test is an identification test for the presence of __. Aromatic amino acid (__ and __) reacts with __ and produce __-colored __.
- aromatic amino acids
- tyrosine; tryptophan
- concentrated nitric acid
- yellow
- nitro derivatives
In the Xanthoproteic test, the benzene ring of __ and the hydroxyl group of __ are easily nitrated into yellow-colored aromatic compounds (__). The addition of NaOH to this solution results in the __ of the phenolic group which produces an __ color.
- tryptophan
- tyrosine
- xanthoproteic acid
- ionization
- orange
