PRECIPITATION REACTIONS OF PROTEINS
PRECIPITATION
REACTIONS OF PROTEINS
Proteins have either a positive
or a negative charge depending on the pH of the solution. If they are treated
with either a metallic salt or strong acid, these charges are neutralised and
the proteins precipitate out. Proteins form colloidal solution in which a shell
or colour of water molecules surround the proteins molecule. If salt solutions
of suitable concentration and charge are added, these ions destroy the shell of
hydration, neutralise the charge on the protein molecule and the proteins
precipitate out. Because of these properties proteins are preciitated out from
their solution.
EXPERIMENT
Egg:
white is the sources of protein used for the following experiments:-
I. Precipitation By Neutralisation and
Dehydration:
Effect
of concentration neutral salt solution:
(i) Half Saturation:
To 3ml of protein solution add
an equal volume of saturated ammonium sulphate solution, a white ppt is formed.
This is precipitation of proteins by half saturation with ammonium sulphate.
(ii) Full Saturation:
To 3ml of protein solution add
solid ammonium sulphate with mixing untill the solution is saturated (i.e.
there should be some undissolved salt in the bottom of the test tube). Let this
stand for 5 minutes. Write your inference.
Albumin being more hydrated than
glubulin, is precipitated by full saturation while globulin is precipitated by
half saturation. This property can be used for separating albumins from
globulins. Higher the molecular weight of a protein, lower the concentration of
salt required for precipitation from solution.
II. Precipitation By Heavy Metal Ions:
When solutions of lead acetate,
mercuric nitrate zinc sulphate & silver nitrate are added to protein
solutions, the cations interact with negatively charged groups on proteins
causing precipitation as metal-Proteinate and in many cases denaturation.
Note:- Raw eg: white is used as an immediate antidote in mercury poisoning
and then an emetic to remove Hg4 ions which are held by albumin.
To 1 ml of protein solution add
5-10 drops of lead acetate solution and observe the protein being precipitated.
Repeat the experiment with solutions of 5% mercuric nitrate, zinc sulphate and
silver nitrate solutions.
III. Precipitation by Alkaloidal
Reagents:
These alkaloidal reagents are
used to get protein free filtrate in the clinical investigation.
To 1ml of protein solution add
few drops 20% sulph salicylic acid. The protein is precipitated. Repeat the
experiment with 1% picric acid, 10% Trichloroacetic acid, phos photungstic acid
and Esbach’s reagent (1gm of picric acid. 2gm of citric acid in 10m1 water).
Esbach’s reagent is used for quantitative determination of albumin in urine.
IV. Precipitation by Strong Mineral
Acid:
This is used for detection of
protein in urine.
Heller’s test
To 3ml of strong HNO3 add drop
by drop of the given protein carefully along the side of the tube. A white ring
of precipitate due to the formation of acid meta-protein is observed.
V. Precipitation by Alcohol:
To 1ml of protein solution add 2ml
of ethanol, precipitation occurs, due to the denaturation of protein.
VI. Heat Coagulation Test:
Take 3/4 test tube of protein.
Hold the test tube in a slanting position and only boil the top layer, observe
for coagulum. The lower half serves as a control. Add 1-3 drops of acetic acid,
persistence of coagulum confirm the presence of protein.
Denaturation is a change in the native
character of the protein brought about by various chemical and physical agents.
A denatured protein becomes less viscous, less soluble and looses its chemical
property. All proteins do not coagulate on heating. Only albumin & globulin
coagulate on heating.
Precipitation reactions are
useful in the separation of serum proteins Deproteinisation of blood or body
fluids for chemical analysis is brought by precipitation reactions.
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