Biochemistry for Psychology Reviewer (2nd - Involves the disruption and possible destruction
of both the 2o and 3o structures, not strong
Semester)
enough to break peptide bonds, 10 remains
Protein Denaturation & Precipitation after denaturation
► Solubility Test
- The solubility of amino acids and proteins is
largely dependent on the pH of the sol’n. The
structural changes in an amino acid or protein
that take place at different pH values alter the
relative solubility of the molecule. In acidic
solutions, both amino and carboxylic groups are
protonated. In basic solutions, both groups are
deprotonated.
- Amino acids are essentially soluble in water.
Their solubilities in water, dilute alkali and
► Heat
dilute acid vary from one one to the other
depending on the structure of their side chains. - Heat can be used to disrupt hydrogen bonds
- When this test is applied to Glycine, Tyrosine, and non-polar hydrophobic interactions.
Glutamic acid and Cysteine. - This occurs because heat increases the kinetic
- Glutamic acid – most soluble energy and causes the molecules to vibrate so
- Followed by Tyrosine due to OH present in its rapidly and violently that the bonds are
side chain, followed Cysteine due to SH group disrupted.
- Least soluble to nonsoluble is glycine being a
► Alcohol
non-polar amino acid.
- Hydrogen bonding occurs between amide OH
► Denaturation of Proteins
groups in the secondary protein structure.
- Involves the disruption and possible destruction - Hydrogen bonding between "side chains"
of both the secondary and tertiary structures. occurs in tertiary protein structure in a variety
- Since denaturation reactions are not strong of amino acid combinations.
enough to break the peptide bonds, the primary - All of these are disrupted by the addition of
structure (sequence of amino acids) remains another alcohol.
the same after a denaturation process. - Alcohol as Denaturant
- Denaturation occurs because the bonding - Max ppt’n occurs at isoelectric point, When
interactions responsible for the secondary allowed to stand in alcohol, protein undergoes
structure (hydrogen bonds and amide bonds) irreversible change and is coagulated (principle
and tertiary structure are disrupted. in the fixing of tissue for biological examination.
- In tertiary structure there are four types of
bonding interactions between "side chains" ► Action of Alcohol
including: hydrogen bonding, salt bridges, - A 75% alcohol solution is used as a disinfectant
disulfide bonds, and non-polar hydrophobic on the skin. This concentration of alcohol is able
interactions. which may be disrupted. 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.
,► Acids and Bases Disrupt Salt Bridges Addition of a neutral salt, such as Ammonium
sulfate, compresses the solvation layer and
- Salt bridges result from the neutralization of an
increases the protein-protein interaction. As the
acid and amine on side chains. salt concentration of a solution is increased,
- The final interaction is Ionic between the more of the bulk water becomes associated
positive Ammonium group and the negative with the ions. As a result, less water is available
Carboxyl group. to take part in the solvation layer around the
- Any combination of the various acidic or basic protein, which exposes hydrophobic parts on
amino acid side chains will have this effect the protein surface.
o Organic acids: Tricholoroacatic acid,
Phosphotungistic acid are frequently used ► Identification of Proteins
as precipitants.
- Proteins are present in the living world,
o Inorganic acids: HNO3 is used for detecting
irrespective of the size of the organism, since
the presence of proteins in urine (Heller’s
they form the structural and functional basis of
test)
cell. Under certain circumstances, it may
► Heavy Metal Salts become necessary to identify the presence of
protein. Isolation of any new compound (e.g.,
- Heavy metal salts act to denature proteins in antiviral principle) needs to be attacked
much the same manner as acids and bases. primarily through identification reactions. Some
- Heavy metal salts usually contain Hg+2, Pb+2 , of the color reactions of proteins which could
Ag+1 Tl+1, Cd+2 and other metals with high be used as identification tests are given below:
atomic weights. Since salts are ionic they
disrupt salt bridges in proteins. ► Protein Electrophoresis
- The reaction of a heavy metal salt with a - Electrophoresis is the controlled motion of
protein usually leads to an insoluble metal charged molecules using an electrochemical
protein salt. cell. As such, electrophoresis apparatuses are
► Heavy Metals Action on Disulfide Bridges constructed of an anode (oxidation half-
reaction) and a cathode (reduction half-
- Heavy metals may also disrupt disulfide bonds reaction) connected by a salt bridge which
because of their high affinity and attraction for maintains electrostatic neutrality.
Sulfur and will also lead to the denaturation of - In protein electrophoresis, a porous solid matrix
proteins. made of polymerized acrylamide
- Disulfide bonds are formed by oxidation of the (polyacrylamide gel) constitutes the salt bridge.
Sulfhydryl groups on cysteine. . Different As depicted, negatively charged molecules
protein chains or loops within a single chain are migrate toward the anode (anions to anode)
held together by the strong covalent disulfide while cations travel to the cathode. Since
bonds. proteins are charged molecules, they are
influenced by electric fields and, depending on
their charge, will migrate toward one of the
- Disruption of Disulfide Bridge electrodes when introduced to the
- Heavy metals like Hg, Ag and Pb precipitates electrochemical system.
protein from alkaline sol’n. Protein behaves like - The ability of a biological molecule to move
acids bec of the COOH present forms insoluble through the porous matrix is known as its
salts with alkali electrophoretic mobility, which is influenced
most dominantly by the charge, size, and shape
► By Neutral Salts of the molecule.
- The precipitation of a protein by neutral salt is
commonly known as salting-out method.
, Lipid Chemistry: Solubility Test - Negative Result: Froth doesn't appear in the
test tube.
► Solubility Test
- Solubility test is the preliminary test which
detects the presence of all lipids. This test
detects the solubility of lipid in various solvents.
To check whether it is miscible or immiscible in
polar or non-polar solvents. ► Sudan IV
- Principle: Solubility test is based on the
property of lipid to dissolve in different - A test used to detect the presence of lipids in a
solvents. Lipids are readily miscible in non-polar solution.
solvents like chloroform, partially soluble in a - Principle: This test is based upon the principle
polar solvent like ethanol and immiscible in a of binding and solubility of lipids to non-polar
polar solvent like water. compounds. As Sudan IV is a non-polar stain,
- Positive Result: Lipids are soluble in non-polar therefore, the lipid will bind with it and retain
solvent i.e. chloroform are partially soluble in the color of the stain and give a red-orange
ethanol which can solubilize upon heating. color. Sudan IV does not stain or bind to the
- Negative Result: Lipids are insoluble in a polar polar compounds.
solvent i.e. water. - Positive Result: Gives red-orange color to the
solution.
- Negative Result: The solution to the color will
remain unchanged.
► Translucent Spot Test
- A translucent spot test is also a preliminary test ► Acrolein Test
for the lipids which can be detected by the
- A test used to detect the presence of glycerol
appearance of a translucent and greasy spot.
and fat.
- Principle: The lipid will not wet the filter paper,
- Principle: This test is based on the “Dehydration
unlike water. The lipid will form a greasy spot as
reaction”, where the water molecules removed
they are having a greasy texture that will
from the glycerol by the addition of reagent
penetrate into the filter paper. In contrast to
potassium hydrogen sulphate. The reaction
lipid, the spot of water will disappear from the
between glycerol and potassium hydrogen
paper whereas the spot of lipid appears as the
sulphate results in the formation of “Acrolein”
“translucent spot.”
that is characterized physically by the release of
- Positive Result: Translucent spot will appear on
the pungent smell.
the filter paper.
- Positive Result: If glycerol present in the sample
- Negative Result: Translucent spot will not
will give a pungent smell.
appear on the filter paper.
- Negative Result: If glycerol is absent in the
sample, it will not produce a pungent smell.
► Saponification Test
► Oxidation of Unsaturated FA
- Positive Result: Froth appears in the test tube.
of both the 2o and 3o structures, not strong
Semester)
enough to break peptide bonds, 10 remains
Protein Denaturation & Precipitation after denaturation
► Solubility Test
- The solubility of amino acids and proteins is
largely dependent on the pH of the sol’n. The
structural changes in an amino acid or protein
that take place at different pH values alter the
relative solubility of the molecule. In acidic
solutions, both amino and carboxylic groups are
protonated. In basic solutions, both groups are
deprotonated.
- Amino acids are essentially soluble in water.
Their solubilities in water, dilute alkali and
► Heat
dilute acid vary from one one to the other
depending on the structure of their side chains. - Heat can be used to disrupt hydrogen bonds
- When this test is applied to Glycine, Tyrosine, and non-polar hydrophobic interactions.
Glutamic acid and Cysteine. - This occurs because heat increases the kinetic
- Glutamic acid – most soluble energy and causes the molecules to vibrate so
- Followed by Tyrosine due to OH present in its rapidly and violently that the bonds are
side chain, followed Cysteine due to SH group disrupted.
- Least soluble to nonsoluble is glycine being a
► Alcohol
non-polar amino acid.
- Hydrogen bonding occurs between amide OH
► Denaturation of Proteins
groups in the secondary protein structure.
- Involves the disruption and possible destruction - Hydrogen bonding between "side chains"
of both the secondary and tertiary structures. occurs in tertiary protein structure in a variety
- Since denaturation reactions are not strong of amino acid combinations.
enough to break the peptide bonds, the primary - All of these are disrupted by the addition of
structure (sequence of amino acids) remains another alcohol.
the same after a denaturation process. - Alcohol as Denaturant
- Denaturation occurs because the bonding - Max ppt’n occurs at isoelectric point, When
interactions responsible for the secondary allowed to stand in alcohol, protein undergoes
structure (hydrogen bonds and amide bonds) irreversible change and is coagulated (principle
and tertiary structure are disrupted. in the fixing of tissue for biological examination.
- In tertiary structure there are four types of
bonding interactions between "side chains" ► Action of Alcohol
including: hydrogen bonding, salt bridges, - A 75% alcohol solution is used as a disinfectant
disulfide bonds, and non-polar hydrophobic on the skin. This concentration of alcohol is able
interactions. which may be disrupted. 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.
,► Acids and Bases Disrupt Salt Bridges Addition of a neutral salt, such as Ammonium
sulfate, compresses the solvation layer and
- Salt bridges result from the neutralization of an
increases the protein-protein interaction. As the
acid and amine on side chains. salt concentration of a solution is increased,
- The final interaction is Ionic between the more of the bulk water becomes associated
positive Ammonium group and the negative with the ions. As a result, less water is available
Carboxyl group. to take part in the solvation layer around the
- Any combination of the various acidic or basic protein, which exposes hydrophobic parts on
amino acid side chains will have this effect the protein surface.
o Organic acids: Tricholoroacatic acid,
Phosphotungistic acid are frequently used ► Identification of Proteins
as precipitants.
- Proteins are present in the living world,
o Inorganic acids: HNO3 is used for detecting
irrespective of the size of the organism, since
the presence of proteins in urine (Heller’s
they form the structural and functional basis of
test)
cell. Under certain circumstances, it may
► Heavy Metal Salts become necessary to identify the presence of
protein. Isolation of any new compound (e.g.,
- Heavy metal salts act to denature proteins in antiviral principle) needs to be attacked
much the same manner as acids and bases. primarily through identification reactions. Some
- Heavy metal salts usually contain Hg+2, Pb+2 , of the color reactions of proteins which could
Ag+1 Tl+1, Cd+2 and other metals with high be used as identification tests are given below:
atomic weights. Since salts are ionic they
disrupt salt bridges in proteins. ► Protein Electrophoresis
- The reaction of a heavy metal salt with a - Electrophoresis is the controlled motion of
protein usually leads to an insoluble metal charged molecules using an electrochemical
protein salt. cell. As such, electrophoresis apparatuses are
► Heavy Metals Action on Disulfide Bridges constructed of an anode (oxidation half-
reaction) and a cathode (reduction half-
- Heavy metals may also disrupt disulfide bonds reaction) connected by a salt bridge which
because of their high affinity and attraction for maintains electrostatic neutrality.
Sulfur and will also lead to the denaturation of - In protein electrophoresis, a porous solid matrix
proteins. made of polymerized acrylamide
- Disulfide bonds are formed by oxidation of the (polyacrylamide gel) constitutes the salt bridge.
Sulfhydryl groups on cysteine. . Different As depicted, negatively charged molecules
protein chains or loops within a single chain are migrate toward the anode (anions to anode)
held together by the strong covalent disulfide while cations travel to the cathode. Since
bonds. proteins are charged molecules, they are
influenced by electric fields and, depending on
their charge, will migrate toward one of the
- Disruption of Disulfide Bridge electrodes when introduced to the
- Heavy metals like Hg, Ag and Pb precipitates electrochemical system.
protein from alkaline sol’n. Protein behaves like - The ability of a biological molecule to move
acids bec of the COOH present forms insoluble through the porous matrix is known as its
salts with alkali electrophoretic mobility, which is influenced
most dominantly by the charge, size, and shape
► By Neutral Salts of the molecule.
- The precipitation of a protein by neutral salt is
commonly known as salting-out method.
, Lipid Chemistry: Solubility Test - Negative Result: Froth doesn't appear in the
test tube.
► Solubility Test
- Solubility test is the preliminary test which
detects the presence of all lipids. This test
detects the solubility of lipid in various solvents.
To check whether it is miscible or immiscible in
polar or non-polar solvents. ► Sudan IV
- Principle: Solubility test is based on the
property of lipid to dissolve in different - A test used to detect the presence of lipids in a
solvents. Lipids are readily miscible in non-polar solution.
solvents like chloroform, partially soluble in a - Principle: This test is based upon the principle
polar solvent like ethanol and immiscible in a of binding and solubility of lipids to non-polar
polar solvent like water. compounds. As Sudan IV is a non-polar stain,
- Positive Result: Lipids are soluble in non-polar therefore, the lipid will bind with it and retain
solvent i.e. chloroform are partially soluble in the color of the stain and give a red-orange
ethanol which can solubilize upon heating. color. Sudan IV does not stain or bind to the
- Negative Result: Lipids are insoluble in a polar polar compounds.
solvent i.e. water. - Positive Result: Gives red-orange color to the
solution.
- Negative Result: The solution to the color will
remain unchanged.
► Translucent Spot Test
- A translucent spot test is also a preliminary test ► Acrolein Test
for the lipids which can be detected by the
- A test used to detect the presence of glycerol
appearance of a translucent and greasy spot.
and fat.
- Principle: The lipid will not wet the filter paper,
- Principle: This test is based on the “Dehydration
unlike water. The lipid will form a greasy spot as
reaction”, where the water molecules removed
they are having a greasy texture that will
from the glycerol by the addition of reagent
penetrate into the filter paper. In contrast to
potassium hydrogen sulphate. The reaction
lipid, the spot of water will disappear from the
between glycerol and potassium hydrogen
paper whereas the spot of lipid appears as the
sulphate results in the formation of “Acrolein”
“translucent spot.”
that is characterized physically by the release of
- Positive Result: Translucent spot will appear on
the pungent smell.
the filter paper.
- Positive Result: If glycerol present in the sample
- Negative Result: Translucent spot will not
will give a pungent smell.
appear on the filter paper.
- Negative Result: If glycerol is absent in the
sample, it will not produce a pungent smell.
► Saponification Test
► Oxidation of Unsaturated FA
- Positive Result: Froth appears in the test tube.
