Experiment III: Analysis Of Proteins

EXPERIMENT III ANALYSIS OF PROTEINS Proteins function in almost all physiological activities of living things viz as cell architecture, catalyst (enzyme), metabolite controller, contractile process, protector (antibodies) and other important compounds in higher organisms. Various molecules Proteins in living organisms have very different structures and folds vary so that they have specific biological activities in cell metabolism. Protein structure is divided into four levels: primary, secondary, tertiary, and quaternary. The four protein structures are differentiated based on a review of the elements and types of chemical bonds involved. The primary structure consists only of one type of bond, namely the covalent bond that connects the amino group and the amino group carboxyl between amino acids or also known as a peptide/amide bond. By Therefore, the primary structure contains information about the amino acid sequence construct a protein and review the basic structure of proteins. Meanwhile the structure secondary involves hydrogen bonding between the carbonyl oxygen and the amide hydrogen (C=O…..HN) from the peptide bond. These hydrogen bonds are formed according to a pattern orderly, such that unique structures are formed such as --helical And--sheets (twodimensional structure). In the higher structure, namely the tertiary structure, the elements secondary structure elements are packaged into a certain shape (three-dimensional structure). This packaging involves various chemical bonds and interactions such as bonding disulfides between cysteine amino acids, hydrogen bonds, ionic interactions between functional groups ionized, hydrophobic and hydrophilic interactions may even exist coordinating covalent bonds as in metalloproteins. All ties and interactions Apart from forming a tertiary structure, this also acts as a stabilizer. Structure the latter, namely the quaternary structure, occurs in some proteins that have more than one subunit. In the quaternary structure there is interaction between the structures Tertiary proteins form aggregates that have certain biological activities. The bond involved are usually non-covalent and mostly hydrophobic interactions between regions non-polar on the surface of protein molecules. Hemoglobin for example, consists of four polypeptide chains (4 subunits), usually two pairs of identical subunits forms hemoglobin tetramer which has a more effective function in transporting oxygen than in the monomer state. Protein denaturation is defined as a state where changes have occurred protein structure that includes three-dimensional shape changes and folds molecules, without involving breaking or damaging the primary structure of the protein. Protein denaturation causes proteins to lose their biological activity and the solubility will decrease so it will easily precipitate. In this experiment you will learn how to identify proteins, and will also observe them physical influences such as temperature, pH and chemicals on protein structure. 1. Biuret reaction Protein solution in a strong base with a few drops of CuSO solution added4runny will form a purple color and this reaction is called the Biuret reaction. Biuret is produced by heating the urea to a temperature of approximately 180oC. The Biuret reaction occurs due to the formation of a Cu complex2+with –CO and –NH groups of the peptide chain in alkaline conditions. Dipeptides and amino acids (excl histidine, serine and tyrosine) did not give a positive reaction to this test. Reactor : - 10% NaOH - 0.1% CuSO4 - Sample: 2% albumin, 2% casein, amino acids Procedure: 1. Into the test tube add 1 mL of sample and 1 mL of 10% NaOH, stir vigorously strong. Add 1 drop of 0.1% CuSO4, stir well. If no color appears add a few more drops of CuSO4until color forms. 2. Into a test tube, add a little urea and heat it until it melts. Cool and watch the smell. Dissolve the cooled urea in top with water, then do as in method (1). Question : 1. What colors and complex compounds are formed? Why should it be avoided advantages of CuSO4? 2.Deposition by Metals Heavy metal cations such as Hg2+, Pb, Cu, Ag, Au, Pt, and others can precipitate proteins in an alkaline environment. Large cations can disrupt ionic interactions namely neutralizing the negative charge in the protein so that denaturation occurs. these ions It can also denature proteins because it reacts with –SH groups to form sulfide. Reactor : - albumin 2% - PbAcetate 2% - HgCl22% - FeCl32% - CuSO42% Procedure: To 1 mL of albumin solution add dropwise the finite metal solution deposition occurs. Pay attention to the changes that occur with each drop. Also pay attention to whether a precipitate forms, and whether the precipitate that forms dissolves returns or increases with the addition of excess reagent. Question : 1. What is the process of protein precipitation with metal? 2. Explain why egg white is used as an antidote for Pb poisoning or Hg! 3. Protein Isoelectric Point Proteins are hydrophilic colloids that are stabilized by charge and protein interactions with solvent. If one of these two factors is removed then the protein Sometimes it can precipitate and if the two factors above are removed then the protein always remains settles. Protein solubility is lowest at its isoelectric point, namely the pH of the solution which causes the number of positive and negative charges in the protein molecule to increase The same. Reactor : - 0.5 % Casein - Acetic Acid 0.1 N and Sodium Acetate 0.1 N - Acetate Buffer pH 6.0: add 10 mL 0.1 N acetic acid to 190 mL 0.1N sodium acetate. - Acetate Buffer pH 5.3: add 29 mL 0.1 N acetic acid to 171 mL 0.1N sodium acetate. - Acetate Buffer pH 5.0: add 59 mL 0.1 N acetic acid to 141 mL 0.1N sodium acetate. - Acetate Buffer pH 4.1: add 147 mL 0.1 N acetic acid to 53 mL 0.1N sodium acetate. - Acetate Buffer pH 3.8: add 176 mL 0.1 N acetic acid to 24 mL 0.1N sodium acetate. Procedure : 1. Into 5 test tubes each add 5 mL of 0.5% casein solution. Next, add acetate buffer pH 6.0 to all tubes; 5.3 ; 5.0 ; 4.1 and 3.8. Shake the mixture well and note the degree of turbidity after: 0 minutes, 10 minutes and 30 minutes. 2. After 30 minutes the entire tube is heated in a boiling water bath for 30 minutes. The fastest formation of sediment/turbidity occurs near isoelectric point of a protein solution. Observe what happens and record it in the table following: