+

Proteins
Qualitative Tests for Proteins
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Proteins

 Proteins are derived from the Greek protas meaning "of primary
importance”)
 Complex, high-molecular-weight molecules
 Biochemical molecules that consists of amino acids joined by
peptide bonds
 Proteins can be hydrolyzed by acids, bases or specific enzymes.
 Proteins are probably the most important class of biochemical
molecules
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Peptide Bond Formation
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Denaturation of Proteins

 Denaturation is the disruption of secondary, tertiary and

quaternary structure of proteins leading to loss of their
biological activity.
 Proteinsdenature when they lose their three-dimensional
structure.
 Proteinsmay be denatured at the secondary, tertiary and
quaternary structural levels, but not at the primary
structural level.
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Levels of Protein Structure:

1- Primary structure:
linear amino acid sequance,strarting from N terminal to C
terminal.
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Levels of Protein Structure:

2- Secondary structure:
 Isthe general three-dimensional form of protein fromed by
hydrogen bonding of amide groups.

Alpha Helix:
 Polypeptide chain is coiled rightly as coil
 The backbone forms the inner part while the side chains extends
outward
 Stabilized by hydrogen bonds of one amino acid and the carbonyl gp
in the 4th amino acid
+ Levels of Protein Structure:
Beta Pleated Sheet :
 Polypeptide chains are aligned side by side with another chain
aligned oppositely
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Levels of Protein Structure:
3- Tertiary structure:
 The entire three-dimensional shape of
the protein. This shape is determined
by the sequence of amino acids
 Havea single
polypeptide chain "backbone" with one
or more
protein secondary structures.
 Formed by hydrophobic interactions,
but hydrogen bonds, ionic interactions,
and disulfide bonds are usually
involved too.
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Levels of Protein Structure:

4- Quaternary structure:
The shape or structure that results
from the union of more than one
protein molecule, usually called
protein subunits in this context,
which function as part of the larger
assembly or protein complex
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Causes of denaturation

Physical factors Chemical factors such

such as heating as strong acid or base
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Characteristics of denatured
proteins are :

1-Loss of function: Most biological proteins lose their biological function when
denatured, for example, enzymes lose their catalytic activity

2- They become less soluble. As a result, they are easily precipitated.

3- Reversibility and irreversibility: In many proteins (unlike egg whites),

denaturation is reversible (the proteins can regain their native state when the
denaturing influence is removed).
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Qualitative Tests for Proteins

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1- Biuret Test

 It is the general test for all proteins.

 Biuret reagent is dilute CuSO4 in strong alkaline medium.

 Alkaline CuSO4 reacts with all compounds containing 2 or more

peptide bonds to give a blue-violet color.

Method:
1 ml of biuret reagent + 1 ml of protein ……mix well>>>> blue-
violet color.
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2- Denaturation by heat and extreme
pH:
 Extreme heating and pH (conc. acids) denature proteins
leading to precipitation of proteins.

Method:
 3ml Protein >>>>>>BWB-10min >>>>>> ppt of protein.
 3mlProtein >>>>>> drops conc.HCL >>>>>> ppt of
protein.
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3- Precipitation of proteins by heavy
metals:
 Proteinsare precipitated in alkaline medium with heavy metals
due to the direct union of cation (Cu++, Ag+, Ba++, Pb++) with
anionic groups of proteins, which are formed in basic medium
 Atalkaline pH 7 and above, proteins are usually negatively
charged so the addition of positively charged ions will neutralize
this charge and the proteins come out of solution (i.e. heavy
metals combine with proteins forming insoluble
metalloproteine).
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At PH > 7
Negativ
Negativ
ely
ely
charged
charged
proteins
proteins

insoluble
metalloprotei
ne

Positevly
Positevly
charge
charge
heavy
heavy
metalsCu++
metalsCu
+, Ag+, Ba+
+, Ag+, Ba+
+, Pb++)
+, Pb++)
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Method:
 Fewdrops of heavy metals + 2ml protein + few drops
10% NaOH>>>>ppt
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4- Precipitation of proteins by acidic
reagent:
 Proteinsare precipitated in acidic medium with some
reagents such as TCA, picric acid and tannic acid due to the
direct union of the anionic group with the cationic groups of
the proteins, which are formed in acidic medium.
-These compounds carry large negative charges which
neutralize the positively charged protein to form insoluble
salt complex with protein.
-The acidic reagents are therefore most effective at acidic
medium where proteins are positively charged.
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At PH < 7
Positive
Positive
ly
ly
charged
charged
proteins
proteins

insoluble salt
complex with
protein

Negati
Negati
evly
evly
charge
charge
Acids
Acids
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Method:
 Few
drops of acidic reagent + 2ml protein >>>slowly add dilute
NaOH and observe the result as the pH increase.
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Practical

 Perform the previous tests on Albumin, Peptone an

gelatine