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Tests for proteins - Biochemistry lab

Tests for proteins is the tests that are used for determine proteins and indicate it form other dietary fuels , we carried out this tests in our biochemistry lab in college of pharmacy - third stage - university of sulaimani .

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Tests for proteins - Biochemistry lab

  2. 2. BiochemistryLab/Proteins Amino acids are among the best known components of living organisms. They are derived from organic acids, There are more than 300 different amino acids described . only 20 commonly occur in almost all proteins. The presence and location of amino acids in the structure of protein molecules is genetically determined. A fragment of the amino acid molecule, composed of the carbon, the α-amino group and the α-carboxyl group is a common structural element of all protein amino acids . At physiological pH (about 7.4), most of the carboxyl groups are dissociated, create anion -COO- , and most of the amino groups bind H+ creating cation -NH3+. Under these conditions, the dominant form of the amino acid is therefore a zwitterion, which has two opposite electric charges. Most of the tests are for the indication of R in the amino acids because most of the amino acids have carboxylic group , amine group and hydrogen . but different R groups . Protein tests are : 1-Ninhydrine test : General test for protein . Indication / Purple or violet complex . 2- Elements of protein : to determine the protein composed of what . Indication / depends on reagent used . 3- Biuret test : to indicate peptide bond . Indication / cupper coordination complex ( violet color ) . 4- Xanthoproteic test : to indicate the aromatic amino acids . Indication / 5- Rosenheim test : test for indole ring . Indication / Brown color ring . 6- Denaturation of protein : involves the destruction of its spatial structures while retaining the primary structure indication / formation of precipitate depends on the reagent used .
  3. 3. BiochemistryLab/Proteins 1-Ninhydrine test : general test for proteins . - ninhydrine consists of 585 amino acids that bonded together by peptide bond .- this test is common in all amino acids because its acts in the basis of protein not amino acids . t reacts with amino acids of theninhydrine is the strong oxidizing agent tha- .purple complexprotein and produces 1-using two test tubes , the first one we adding albumin and the second one adding fructose . 2-we add 5 drops of ninhydrine for both test tubes. 3-put them into the water bath for 5 mins 4- determine wether the purple complex is formed or not . 2- Elements of protein : this test is applied to make sure that the amino acid of the protein contain carbon atom , amino group , carboxylic group and also to indicate sulfur . -To determine the carbon , put the sample in test tube and heat it on the hot plate , the Black color is observed ( black burned color ) which it means this sample contain carbon atom . - To determine the oxygen or carboxylic group in the sample , put your sample in the test tube and heat it on the hot plate , the bubbles or vapor is formed inside the test tube which it means the sample contains oxygen or carboxylic group .
  4. 4. BiochemistryLab/Proteins - To determine the amino group , add a litmus paper into the sample in test tube ( red litmus paper is changed to blue or blue litmus paper remains blue ) means that the sample contain Nitrogen or amino group . - To determine the Sulfur , put filter paper into lead acetate then into the sample (albumin) , the black ppt is formed (PbS) which it means the side chain of the amino acid contains sulfur . Note / Many amino acid side chains contain sulfur , that’s why we do this procedure . 3-Biuret Test : this test is applied to indicate peptide bond within protein . Amino acids are building blocks of the proteins that are linked together by peptide bonds . -Reagents in this test are CuSo4 and NaOH. -CuSo4 ( Chelating agent ) is the source of CU++ -NaOH is to raise the Ph of the medium and reach the solution to alkaline level . the aim of this alkaline solution is for the reaction to occurs. , Cu++ions will formwhen peptide bonds are present in the alkaline solution- from peptide bond.trogen atomscoordination complex with four ni -As the number of peptide bond increase , the more intense the change . -This test gives positive result when 2 or more than 2 peptide bond available. If you do this test for a single amino acid , it will give you negative result . Procedure : Add 30 drops oof albumin into test tube . Then add 5 drops of CusO4 And then 10 drops of NaOH .
  5. 5. BiochemistryLab/Proteins 4- Xanthoproteic test : Test for aromatic amino acid . Aromatic amino acids are L-tyrosin , Tryptophan and Phenyl Alanine . -Reagents used are HNO3 and NaOH . HNO3 is used to for nitration to react with the ring .- -If the reaction occurred the xanthoproteic acid or nitrogen derivate is formed which it has yellow color. -Then NaOH is used to produce a salt ( Orange color salt ). Procedure : Add 30 drops of albumin into test tube . Add 5 drops of nitric acid then heat and shake ( in order for the solution not to coagulate.) Finally add 20 drops of NaOH. 5- Rosenheim Test : Test for indole ring . -Specific test for tryptophan , because tryptophan is the only amino acid that contain indole test . Reagents used are F3Cl3 , CH2O ( formaldehyde ) and H2SO4- FeCl3 gives the color to the solution- -CH2O breaks the bond to differentiate amino acids .
  6. 6. BiochemistryLab/Proteins Until H2SO4 is added , the solution doesn’t show any brown ring .- -H2SO4 causes the formation of two layer in the solution , also brown ring is seen between these two ring . Above layer is protein and the lower layer is H2SO4 .- -Note that this brown ring is not the indole ring , its just a product of the reaction which means the amino acid contain indole ring. Procedure : Add 5 drops of CH2O and 3 drops of FeCl3 Mix the these two reagents together which will form rosenheim reagent. Then add 30 drops of albumin and then shake . until here we doesn’t have any product or result . Add 20 drops of H2SO4 drop by drop ( DON’T SHAKE ) then observe the formation of the colored ring ( brown ring ). 6- Denaturation of Protein : involves the destruction of its spatial structures ( secondary & tertiary structure of protein ) while retaining the primary structure because this reaction is not strong enough to break the peptide bond between amino acids . This reaction doesn’t break peptide bonds , it just breaks the hydrogen and covalent bonds .
  7. 7. BiochemistryLab/Proteins Denaturation of protein is applied by : 1-Heating : 15 drops of albumin is added to a test tube then put the test tube on the hot plate , the result will be a white ppt. the heating causes to destroy the structure of protein this is not about charge . 2-Strong Acid : 15 drops of albumin is added to test tube and then HNO3 or H2SO4 is added , then the white ppt. is formed If HNO3 is used , the test called Heller's Test . 3- Alkaloidal reagent : such as Picric acid , the alkaloidal reagent has a negative charge that will react with the positive part of the amino acid ( NH3+ ) And neutralize the protein , reaches the isoelectric point in which the net charge equals to zero . Finally yellow ppt. will form. 4- Heavy metals : such as lead acetate , ferric chloride , cupper sulfate . Heavy metals are positively charged and this will react negative part of amino acid ( COO- ) and neutralize the protein , reaches isoelectric point . Lead Acetate ( Pb(Ch3coo) ) will form white ppt. Ferric chloride ( Fecl3 ) will form yellow to brownish ppt. Cupper sulfate ( Cuso4 ) will form blue ppts . and second one containtyrosinif you have two samples , one contain-1 how you can differentiate ?tryptophan 2-Formaldehyde is used in which test and why ? 3- how you can indicate aromatic amino acids from other amino acids ? November9, 2017 , Thursday Bryar R. Aliruss
  8. 8. BiochemistryLab/Proteins