protein

• 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.

− C− NH−

• =
• O
• 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 + RCH(NH2) COOH

Triketohydrindene hydrate (oxidizing agent)

RCHO + NH3 + CO2 + reduced Ninhydrin

General amino acid

Reduced Ninhydrin + NH3 + Ninhydrin

blue-violet complex
• The librated NH3 reacts with the hydrindantin (reduced ninhydrin) with a second molecule of Ninhydrin to produce a blue ̶ violet colour complex.

( hydrindantin)

ninhydrin amino acid hydrindantin

− 2 H2O

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

White ppt.

Cu(OH)2

Excess

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

Na2S + pd (CH3COO)2

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

Cystine

• 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.

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





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.

Procedure:

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.

Arginine

Guanidine


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 α- Naphthol mixed again then add 2-6 drops of bromine water, mix very well a bright red colour appear indicate the presence of arginine.

Note

• Excess of bromine water in this test should be avoided otherwise declorization of colour will take place.
• control tube should be done using H2O alone (Blank) since alpha-Naphthal give colour with bromine.
• All known protein contain Arginine in their structure this test could be regarded as general test for proteins.

protein can be classified into:-

1. Simple protein like :
• Albumin & Globulin.
• Scleroprotein .
2. Conjugated protein:
• Phosphoprotein (Casein).
• Glycoprotein (Mucine).


CLASSIFICATION OF PROTEIN

• simple protein

• 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
• 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.