Biochemistry Lab 2: Protein & Specific Tests PDF

protein  Protins are high molecular weight molecules; they are polymers of amino acid. There are two types of proteins: 1- Simple proteins which contain only amino acids. 2. Complex proteins (Conjugated proteins): which contain in addition to amino acids non- amino acids such as h...

protein  Protins are high molecular weight molecules; they are polymers of amino acid. There are two types of proteins: 1- Simple proteins which contain only amino acids. 2. Complex proteins (Conjugated proteins): which contain in addition to amino acids non- amino acids such as heme, vitamin , or lipid (lipoproteins), or carbohydrates(Glycoprotins). Amino acids contain both amino and carboxylic functional groups. Although about 300 amino acids occur in nature, only 20 of these occur in proteins. 1. Source of proteins: Exogenous: take with the diet. Endogenous: synthesized inside the human body.  In healthy adults, the total amount of protein in the body remains constant, because the rate of protein synthesis is equal to that is degraded. So the protein turnover, leads to the hydrolysis and resynthesize of 300 to 400 g of body proteins each day.  Proteins are polymers of amino acids ,they consist of long chains of amino acids linked together by peptide bonds.  Proteins are very important biological molecules. The main functions of protein: Enzymes are proteins that catalyze biological reactions. DNA polymerase, amylase transport proteins such as hemoglobin. storage proteins. Egg albumin  structural proteins: collagen, keratin  contractile proteins: actin, myosin. protective proteins: antibodies. Hormones: insulin. Proteins can be broken down by differents chemical and enzymatic methods into smaller and smaller fragments to the final products are the amino acids.  An amino acid is a molecule that contains an amino group and a carboxyl group in the same molecule. The amino acids that are found in proteins are α- amino acids, which means that the amino group is linked to the alpha carbon. The amino acid side chains can be classified based on whether they are non polar, polar, acidic, or basic. Since proteins are biological molecules, they are usually found in a neutral solution that is buffered at pH 7.0 to 7.4. In this condition, the acidic and basic groups on the amino acids will be ionized. Most amino acids remain as “zwitterions” at pH 7. There are 20 amino acids that differ from each other only in the identity of the side chain attached to the alpha-carbon. Amino acids of acidic R groups (negative charge) Amino acids of basic R groups (posative charge) Amino acids can be linked together the carboxyl group on one amino acid can react with the amino group on another amino acid, forming an amide. Therefore, the bonds linking together the amino acid residues are amide bonds, but in proteins they are called peptide bonds.  Biuret Test - chemical test for detecting presence of peptide bonds in the presence of peptides. -The reaction in this test involves the chelate complex formation of the proteins with Cu+2ions in a strongly alkaline solution. -Apply this test to gelatin, casein and albumin - Positive visible result: violet This reaction can be used to assess the concentration of proteins. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Bee Lambert low. The Biuret reagent is made of KOH and hydrated copper(II) sulfate, together with potassium sodium tartrate. Potassium sodium tartrate is added to complex and stabilize the cupric ions. The reagent turns from blue to violet in the presence of protein, blue to pink when combined with short-chain polypeptide  Hydrated Copper sulfate. This provides the Cu (II) ions which form the chelate complex. Cu (II) ions give the reagent its characteristic blue color. Potassium hydroxide solution does not participate in the reaction but provides the alkaline medium. Potassium sodium tartrate stabilizes the chelate complex.  The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue to violet complex that absorbs light at 540nm. Thus, the biuret reaction is the basis for a simple and rapid colorimetric test for quantitatively determining of total protein concentration.  Ninhydrin It is a common reagent for visualizing spots of amino acids that have been separated by chromatography or electrophoresis. Free amino groups will react with the ninhydrin reagent to yield a blue -purple complex solution. Ninhydrin produces blue-purple dye regardless of the structure of the original amino acid. The side chain of the amino acid is lost as an aldehyde. Mechanism : when amino acids with a free α-NH2 group are treated with ninhydrin solution, performing the oxidation of α-amino acids and decomposition into aldehyde, CO2 and NH3, ninhydrin is becoming reduced; then the two ninhydrin molecules are bound by N derived from the α- NH2 group. Amino acid proline give yellow color with ninhydrin reagent sol because it doesn’t contain free amino group. Some proteins also give a positive test with ninhydrin. Specific reactions for individual amino acids  Millon’s Test  It is specific for tyrosine, the only amino acid that contain a phenol group on which a hydroxyl group is attached.  Millon’s reagent contains mercury dissolved in concentrated HNO3.  Red precipitate is a positive test result for phenols in the sample.  Millon’s chemical reaction: Nitrophenols, formed from tyrosine by the action of nitric acid.  Then the nitrated tyrosine complexes mercury ions in the solution to form red solution positive results.  The red color is probably due to a mercury salt of nitrated tyrosine.  The Millon reaction does not occur with gelatin, because it does not contain tyrosine. Millon’s Reagent is prepared by dissolving 5 g each of HgNO3 and Hg(NO3)2 in 100 ml of dil. HNO3. Some tyrosine containing proteins will give in a white precipitate ,which would turn red when heated Note :  that any compound with a phenol group will yield a positive test sach as. Salicylic acid  MILLON'S REAGENT IS HIGHLY TOXIC HIGHLY AND CORROSIVE