COLOR REACTIONS OF PROTEINS:

 A protein tests for a particular functional group or structural component present in a protein or
in a particular amino acid/s found in proteins.

Why is different test needed to determine protein present in a solution?

 Since protein is a complex structure, there is no definite test for determination of protein.
Different test is needed to determine different functional group present in a particular solution.
Different Color Reactions for Proteins:
1. Biuret test:
 Principle:
 The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in proteins to
form a blue to violet complex known as the “biuret complex”.
 Two peptide bonds at least are required for the formation of this complex.
2. Xanthoproteic Test
 Principle:
 The reaction gives yellow color upon heating as the result of the formation of nitro-
derivatives of the compounds which contain a benzene-ring such as tyrosine or aromatic
rings such as tryptophan and phenylalanine.
 On adding alkali (NaOH) to these nitro derivative salts, the color change from yellow to
orange.
3. Millon’s Test
 Principle:
 The reaction serves as a test for presence of phenolic compounds such as tyrosine.
 Millon’s reagent is composed of mercuric nitrate dissolved in nitric acid.
 A red solution or precipitate that forms in heating is an indication that tyrosine is present.
4. Hopkin’s-Cole Reaction
 Principle:
 The violet ring color is produced is due to the formation of a compound from the glyoxylic
acid in the regent and tryptophan in the protein.
 Hopkins-Cole reagent (magnesium salt of oxalic acid) gives positive results with proteins
containing the essential amino acid “tryptophan” indicating a high nutritive value.
5. Ninhydrin Test
 Principle:
 This test is a general test and thus given by all amino acids. This test is due to a reaction
between alpha amino group of free amino acid and ninhydrin.
 Ninhydrin is a powerful oxidizing agent and its presence, amino acid undergoes oxidative
deamination liberating ammonia, CO2, a corresponding aldehyde and reduced form of
ninhydrin (hydrindantin).
 The NH3 formed from an amino group reacts with another molecule of ninhydrin and is
reduced product (hydrindatin) to give a blue substance diketohydrin (Ruhemanns complex).
 However, in case of amino acid like proline and hydroxyproline, a different product having a
bright yellow color is formed.
 Asparagine, which has a free amide group, reacts to give a brown colored product.
PRECIPITATION REACTON OF PROTEINS:
 Proteins form colloidal systems. Their solubility depends on their electrical charge and the layer
of hydration around them. Hence, proteins can be precipitated by dehydration and
neutralization of electrical charge.

1. Heller’s Test
 Chemical test that shows that strong acids cause the denaturation of precipitated proteins.
Concentrated nitric acid is added to a protein solution from the side of the test tube to form two
layers. A white ring appears between the two layers if the test is positive.
 Heller's test is commonly used to test for the presence of proteins in urine.
2. Robert’s Test
 Robert's Test uses HNO3 and MgSO4. The positive test is appearance of white ring at the
junction of the reagent and the test sample.

DENATURATION OF PROTEINS:
 Denaturation of proteins involves the disruption and possible destruction of both the secondary
and tertiary structures. Since denaturation reactions are not strong enough to break the peptide
bonds, the primary structure (sequence of amino acids) remains the same after a denaturation
process. Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it
into a random shape.

WHY DOES DENATURATION OCCURS?

 Denaturation occurs because the bonding interactions responsible for the secondary structure
(hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are
four types of bonding interactions between "side chains" including: hydrogen bonding, salt
bridges, disulfide bonds, and non-polar hydrophobic interactions. which may be disrupted.
Therefore, a variety of reagents and conditions can cause denaturation. The most common
observation in the denaturation process is the precipitation or coagulation of the protein.

DENATURATION BY HEAT:
 Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions. This
occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly
and violently that the bonds are disrupted. The proteins in eggs denature and coagulate during
cooking. Other foods are cooked to denature the proteins to make it easier for enzymes to
digest them. Medical supplies and instruments are sterilized by heating to denature proteins in
bacteria and thus destroy the bacteria.

ALCOHOL DISRUPTS HYDROGEN BONDING:

 Hydrogen bonding occurs between amide groups in the secondary protein structure. Hydrogen
bonding between "side chains" occurs in tertiary protein structure in a variety of amino acid
combinations. All of these are disrupted by the addition of another alcohol.
  A 70% alcohol solution is used as a disinfectant on the skin. This concentration of alcohol is able
to penetrate the bacterial cell wall and denature the proteins and enzymes inside of the cell. A
95% alcohol solution merely coagulates the protein on the outside of the cell wall and prevents
any alcohol from entering the cell. Alcohol denatures proteins by disrupting the side chain
intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the new
alcohol molecule and the protein side chains.

ACIDS AND BASES DISRUPT SALT BRIDGES:

PRECIPITATION BY ALKALOIDAL REAGENTS:
 Organic acids carry a large negative charge which neutralize positively charged protein to form
an insoluble salt. The acidic reagents are most effective at acid pH value where proteins are
positively charged. The precipitation of proteins by this method is irreversible.
 Salt bridges result from the neutralization of an acid and amine on side chains. Review reaction.
The final interaction is ionic between the positive ammonium group and the negative acid
group. Any combination of the various acidic or amine amino acid side chains will have this
effect.
 As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type
of double replacement reaction occurs where the positive and negative ions in the salt change
partners with the positive and negative ions in the new acid or base added. This reaction occurs
in the digestive system, when the acidic gastric juices cause the curdling (coagulating) of milk.

HEAVY METAL SALTS DISRUPT DISULFIDE BONDS:

 Heavy metals may also disrupt disulfide bonds because of their high affinity and attraction for
sulfur and will also lead to the denaturation of proteins.
 At pH 7 and above, proteins are usually negatively charged, the positively charged metal ions
neutralize the charges of protein causing precipitation of the protein. Precipitation by heavy
metals is therefore most effective at neutral to slightly alkaline pH value.

REDUCING AGENTS DISRUPT DISULFIDE BONDS:

 Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. Review reaction.
Different protein chains or loops within a single chain are held together by the strong covalent
disulfide bonds. Both of these examples are exhibited by the insulin in the graphic on the left.
 If oxidizing agents cause the formation of a disulfide bond, then reducing agents, of course, act
on any disulfide bonds to split it apart. Reducing agents add hydrogen atoms to make the thiol
group, -SH.

METAPROTEINS
 Nondescript term for a derived protein obtained by the action of acids or alkalis, soluble in weak
acids or alkalis but insoluble in neutral solutions; e.g., albuminate.
Evaluation Answers:
1. What protein is soluble in plain water but not soluble in salt water?
o Globulin
2. Can albumin be separated from solution by filtering through filter paper?
o No
3. What kind of poisoning uses egg albumin as an antidote?
o Metal poisoning
4. What test is used in determination of proteins in urine?
o Heller’s test
5. What functional group gives yellow color to Xanthoproteic test?
o Phenyl group/aromatic benzene ring
6. What colors indicate a positive result to Millon’s test?
o White precipitate to reddish brown
7. Give the two functional groups that are contained in an amino acid.
o Carboxylic group
o Amine group