Transcript bath and copper

PROTEINS
• Proteins are a complex nitrogenous group with
high molecular weight .It consist of a large number
of amino acid connected together with a special
bond known as peptied bond.
=
O
− C− NH−
• The difference between protein and carbohydrate
is that protein contain nitrogen in addition of C , H
and O.
Characters of proteins:
• All proteins give a number of a colour reaction due to the
existence of certain amino acids in the protein molecule or
to special chemical group associated with molecular
structure of protein.
• All proteins form colloidal solutions which are usually
opalescent and can be precipitated by variety of reagents.
Elements present in proteins
• All proteins contain N,C,H,O and small quantity of : sulfur,
iodine and phosphate.
Procedure:
In to a dry clean test tube place a small quantity powder of
albumin. Place a piece of moistened red litmus paper on the mouth of
the test tube and heat until we get a smell of burning hair.
1. The red litmus paper will change to blue colour due to evolution of
ammonia from heating protein indicating the presence of N .
2. Moisture on the part of the inside of the test tube due to the
condensation of water and indicate the presence of H and O.
3. Charring of the heated protein indicate the presence of C.
Ninhydrin Test:
• It is the general test for protein and for all products of protein
hydrolysis including amino acid.
• The Ninhydrin test is used to detect the presence of alpha amino acids
and proteins that contain free amino groups, (-NH2)
• Ninhydrine is powerful oxidizing agent which oxidize amino acid
according to the following general equation.
Ninhydrin
+
Triketohydrindene hydrate
(oxidizing agent)
RCH(NH2) COOH
General
amino acid
RCHO + NH3 + CO2 + reduced Ninhydrin
( hydrindantin)
• The librated NH3 reacts with the hydrindantin (reduced
ninhydrin) with a second molecule of Ninhydrin to produce
a blue ̶ violet colour complex.
Reduced Ninhydrin + NH3 + Ninhydrin
blue-violet complex
− 2 H2O
ninhydrin
amino acid
hydrindantin
Procedure:
In a clean two test tube put in the first one 5 drops of
diluted albumin and in the second 5 drops of a.a. Then add to
each test tube 2 drops of ninhydrin solution, mixed very well
then put the two test tube in to a boiling water bath for 3 min.
a blue-violet colour complex will form indicating the presence
of amino acid.
Note:
• Proline and hydroxy proline which lack an amino group,
yield a yellow colour with Ninhydrin test, which contains a
secondary amino acid.
• Some colour test are specific for certain amino acids.
General colour test of protein
The most useful general test of protein are Biuret and
Ninhydrin test.
Biuret Test:
Is commonly used to detect the presence of proteins and
peptiedes by treating a sample with an alkaline solution of
dilute copper sulfate to yield a pink-violet to purple-violet
colour. At least two peptide bonds (tripeptide) are required
for positive test Biuret. The intensity of the colour complex
produced is directly proportional to the number peptied bond
present.
• Biuret reagent is CuSO4 in alkaline NaOH.
Procedure:
Add 5 drops of dilute protein solution add, 5 drops of 10%
of NaOH, mixed and then add 2 drops of 0.5% CuSO4
solution. Compare with a blank tube containing 5 drops of
water and the same amount of NaOH and CuSO4. Describe
any colour change that occurred.
Note:
• Excess copper sulphate should be avoided because copper
hydroxide will be form which has a white ppt. interfering with
the violet or blue colour of Biuret reaction.
CuSO4 + NaOH
Excess
Cu(OH)2
White ppt.
The Millon's Test
• Indicate the presence of Tyrosine when the
phenolic group of tyrosine reacts with reagent a mixture
of (Hg++, Hg2++, HNO3 and HNO2)
1
Tyrosine
2
Nitrous acid
Nitrosophenol
Procedure:
In clean test tube put 10 drops of albumin solution then add 2
drops of Millon's reagent, mixed very well then put the tube in
a boiling water path for (2-5) min. pink-red ppt. will be form
indicating the presence of tyrosine.
Note:
In case of using protein (albumin egg) a red ppt. will be form
because mercuric present in Millon′s reagent will ppt. the
protein, but in case of using tyrosine a red colour solution will
be form.
Test for cysteine or cystine
• The lead acetate test for sulfur in cysteine and cystine produces
a brown to black colour when proteins or peptiedes containing
these
amino acids are heated with an alkaline solution of lead
acetate
resulting the formation of lead sulfide PbS as black ppt.
• The sulfar present as organic form in the amino acid, will change to
inorganic form as Na2S by heating with (40% NaOH), then the lead
acetate solution added a brown to black ppt. of PbS will form
indicating the presence of cysteine and cystine.
Cysteine – SH + 40% NaOH
Na2S + pd (CH3COO)2
Na2S
PdS + CH3COONa
Black ppt.
Procedure :
In clean test tube put 10 drops of albumine then add
10 drops of 40% NaOH, mixed very well then put the
test tube in a boiling water bath for 10 min.(this will
change the sulfur in cysteine from organic to inorganic
form), then add 2 drops of lead acetate, then put the
test tube into a boiling water path for 5 min. a black or
brown ppt. will be form that indicate the presence of
cysteine.
• Cysteine is a monomer and Cystine is a dimmer, which is
formed by 2 cysteine molecules and is more stable than
cysteine, but may not be absorbed as well.
Cysteine
Cystine
Hopkin's Cole Test
• Hopkin's Cole is a specific test for tryptophan, the only
amino acid containing an indole group. results in the formation
of purple ring when a solution containing a mixture of
Hopkin's cole reagent layered over concentrated sulfuric acid.
Structural formula of indole
• The Hopkin's-cole reagent containing of glacial acetic
acid and concentrated H2SO4 .
•The strong acid H2SO4 oxidize the glyoxyl group
(HCO-COH) in glacial acetic acid as impurity to
glyoxilic acid (C2H2O3).
• Then the acid condense with tryptophane the presence
of H2SO4 forming purple colour complex as a ring
which indicate the presence of tryptophane.
L-Tryptophan
Procedure:
In clean test tube put 10 drops of glacial acetic acid then add 10 drops of
tryptophane, mixed very well then add 10 drops of cone. H2SO4 on the
side of the test tube, purple colour complex as a ring appear that is
indicate the presence of Tryptophane.
The Sakaguchi Test
The Sakaguchi test is a specific qualitative test for the
detection of a specific type of protein with the amino acid
containing the guanidinium group,this test is used to detect
the presence of arginine.
Guanidine
Arginine
Principle
• Arginine reacts with α-Naphthol in the presence of
alkalin medium, the product will oxidize with bromine to
form red colour complex.
Sodium hypobromite (NaOBr) + α- Naphthol + NH2 group of
guanidine part of arginine (H2N-C-NH)
NH
Red colour complex alkaline medium
Procedure
In clean test tube put 10 drops of albumin then add 10 drops of
(10 % NaOH), mixed very well then add 2-3 drops of αNaphthol mixed again then add 2-6 drops of bromine water,
mix very well a bright red colour appear indicate the presence
of arginine.
Note
1. Excess of bromine water in this test should be avoided
otherwise declorization of colour will take place.
2. control tube should be done using H2O alone (Blank) since
alpha-Naphthal give colour with bromine.
3. All known protein contain Arginine in their structure this
test could be regarded as general test for proteins.
CLASSIFICATION OF PROTEIN
protein can be classified into:1. Simple protein like :
a. Albumin & Globulin.
b. Scleroprotein .
2. Conjugated protein:
a. Phosphoprotein (Casein).
b. Glycoprotein (Mucine).
1. simple protein
A. Test for albumin & globulin
1. Heat coagulation the main character of this kind of protein
is heat coagulation
Procedure:in clean test tube put 10 drops of protein solution, then heat
directly coagulate will form on the side of the test tube.
2. Separation between albumin & globulin.
• Globulin is too large & high molecular weight than albumin
molecule. According to that globulin is more easily precipitate
than albumin.
• To separated alb. & glob. using different conc. of ammonium
sulphate, glob. will ppt. with 50% of ammonium sulphate,
alb. will ppt. with 100% of ammonium sulphate.
• Both alb. & glob. can ppt. in 100% of ammonium sulphate.
B. Scleroprotein
• Scleroproteins , or fibrous proteins, constitute one of the
three main classes of proteins, alongside globular proteins
and membrane proteins.
• Scleroproteins that is fibrous and insoluble in water, serving
a protective or supportive function in the body. Also called
albuminoid
• Keratin, Collagen, Elastin, and Fibroin are all scleroproteins
• The roles of such proteins include protection and
support, forming connective tissue, tendons, bone
matrices, and muscle fiber.
• Protein which contain a sulfur like cysteine or cystine.
• It is a simple protein as skeletal tissues like : skin, bone
,
hair & nail.
Procedure:• In a clean test tube put apiece of hair, added 15 drops of
40% NaOH, then put the test tube in a boiling water bath
for 5 min (to convert organic sulfur to inorganic form).
• Cool the solution, then added 4 drops of lead acetate and
heat again, black or brown ppt. will be form that indicate
the presence of sulfur as lead sulfide (Pbs).
2.Conjagated protein
A. phosphoprotein:(protein + phosphorous)
• Casein in the milk is the best example for this kind of protein.
• To separate casein from milk by adding dilute acetic acid
maximum precipitate will from near isoelectric point at pH
4.55 (acidic pH).
Principle of the test:
• As we know phosphorous present in casein as organic form
so we change the organic phosphorous to inorganic form by
the presence of nitric acid (conc.HNO3), then treat the result
with ammonium molybdate reagent a yellow ppt. will be
appear that indicate the presence of phosphorous (p), which
mean the presence of Casein.
Procedure:In a clean test tube put 10 drops of casein, then add 2 drops of
conc.HNO3 mixed very well then put the test tube in a boiling
water bath for 5 min.(to convert organic phosphorous to
inorganic form), then add 4 drops of ammonium molybdate
solution, a yellow ppt. will be appear that indicate the presence
of phosphorous (P), it presence of Casein.
B. Glycoprotein
• Glycoproteins are proteins that contain oligosaccharide chains
(glycans) covalently attached to polypeptide side-chains
• It is a conjugated protein (protein + carbohydrate).
• This type has no reducing property because it is lost this
property during the attachment with protein molecule so it give
negative (-ve) with Benedict test. But after hydrolysis it give
(+ve) with Benedict test.
• The main example of Glycoprotein is ″Mucin″ , which present
in saliva.
• The special test for this kind of protein is Molish test.