[Music]

friends

in this video we will be doing the

precipitation reaction of proteins

simple proteins especially the globular

proteins like albumin

dissolves in water to form colloid it

forms the lyophilic colloid to know more

about colloids

you can watch one of my previous video i

will put a link to it in the description

since the stability of lyophilic

colloids are dependent on the water

molecules of solvation and charge

any factor which neutralizes the charge

or removes the water of solvation can

therefore cause precipitation of

proteins

some of the practical aspects of

precipitation of proteins

include the identification of protein

qualitatively from a sample of urine

or for purification of enzymes and other

proteins usually start by precipitation

from

solution there are different methods of

precipitation

let us see them one by one the first one

is the precipitation by salt

that is the saturation test we have a

half saturation and a full saturation

test

when an inorganic salt like ammonium

sulphate is added to a solution of

protein

it decreases the concentration of water

molecules available for stabilizing the

protein solution

and it causes it to precipitate out

known as salting out

albumin tenaciously holds a large number

of molecules of water therefore needs a

much higher concentration of salt to get

precipitated out than

globulin first of all we will be

performing the half saturation test

here we take three milliliters of the

protein solution in a test tube

into that we add three milliliters of

saturated ammonium sulphate solution

after adding the ammonium sulphate

solution we mix it well

we can see that the solution has turned

turbid

but no visible precipitation has taken

place

so let us say that the solution is now a

parallelism

next we perform the full saturation test

for that we first take 3ml of the

protein solution

and into that we add solid ammonium

sulphate crystals

we add the solid ammonium sulphate until

the solution becomes saturated

here i added about half a gram of

ammonium sulphate

after mixing up the solid ammonium

sulphate you can already see a thick

white precipitate settling down the

bottom of the test tube

okay so we now have two solutions now we

filter both the solutions to remove the

precipitate and collect the filtrate

you can definitely make out too much

precipitate in the right sided filter

paper

which was the solution from the full

saturation test now with the filtrate

solution we perform the bio earth test

that is a test for proteins we add an

equal volume of 40

sodium hydroxide and two to three drops

of one percentage copper sulfate

you can see that the beautiful bioret

has formed in the half saturation test

tube

and in the full saturation test tube we

again perform the bioreactors

you can see that there is no formation

of biology indicating

there is no proteins present in it this

is because

in half saturation test only the

globulins got

splitted out and albumins did not come

out of the solution

in the second test solid ammonium

sulphate was added

which used up all the water molecules

including the one which

were used by albumin so the filter

didn't have

protein so the bioretest was negative

now we move on to the second test that

is the isoelectric precipitation so

first of all we need to know what is

isoelectric ph

the ph at which the molecules of a

protein bear

no net charge is called its isoelectric

ph

the isoelectric ph varies with different

proteins and proteins have

minimum solubility at their isoelectric

point

many proteins are precipitated from the

solution
by adjusting the ph close to their

isoelectric point

by addition of an acid or an alkali here

we have taken three milliliters of

albumin the isoelectric ph of albumin is

4.7

then we add one to two drops of

bromocrystal green indicator

which has a ph range of 3.8 to 5.4

so that means when there is a change in

ph

above the isoelectric ph there will be a

color change for this indicator

then we slowly start adding one

percentage acetic

acid initially the ph was basic and the

color was blue

on adding the acetic acid it changed

acidic and the color turned green

and you can see that when it closed the

isoelectric ph

the precipitation took place

now let us see the precipitation by

organic solvents

proteins in water generally forms

hydrogen bonds

organic solvents like acetone ether or

ethanol when added to a protein solution

in water

reduces the concentration of water

molecules available for keeping the

proteins in solution

thereby destroying the hydrogen bonds

and causing precipitation

here into two milliliters of the sample

protein solution

i added about 1 ml of ethanol on adding

the ethyl alcohol you can see the

precipitation has already taken place

now we see the precipitation by heavy

metal ions

proteins generally in solution that is

alkaline

has negatively charged anions positively

charged heavy metal cations then binds

with this negatively charged protein

causing precipitation salts of iron

copper zinc lead cadmium and mercury are

toxic

because they tend to precipitate out the

normal proteins present in the

gastrointestinal tract

here you can see into protein solution i

added

10 percentage mercuric nitride solution

on adding the mercuric nitrate solution

into that

there is formation of a white

precipitate
that is the precipitated proteins now we

see the precipitation by

strong mineral acids strong mineral

acids can cause coagulation of proteins

the coagulation using concentrated

nitric acid known as the halos nitric

acid test is used to identify the

presence of protein in urine

to 2 ml of sample protein solution i

added about

2 ml of concentrated nitric acid and

that is the precipitated proteins

now we do the asbestos test so what is

sbac's reagent

aspect reagent is composed of picric

acid citric acid and water

in the ratio of 1 is to 2 is to 97

the reagent being anionic neutralizes

the positive charge of proteins and

precipitation takes place

here on adding about 1 ml of the asbax

reagent

into the protein sample solution you can

see the precipitation taken place

i would like to take this opportunity to

thank my patreon supporters who have

financially supported me

so that i could get the materials

required for doing all these experiments

including this one thank you thank you

so much for watching this video

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