MODULE 8 PROTEIN COMPOUND TEST Basic Theory Protein comes from the words protos or proteos which means first or main. Proteins in cells functions as the main substance in the formation and growth of the body, it can also be used as source of energy if the body lacks carbohydrates and fats. Through hydrolysis by acids or enzymes, Protein will produce amino acids. Based on their structure, proteins are classified into simple proteins and combined proteins. Simple proteins, consisting only of simple molecules (for example fiber protein and globular protein),while combined proteins consist of proteins and prosthetic groups and consist of carbohydrates, fats, or nucleic acids. Protein has meaning for the body if the protein can carry out biochemical activities supports the body's needs. This activity depends on the structure and conformation of the protein molecule. If protein conformation changes, for example by changes in temperature, pH, or reactions with other compounds, metal ions, then the biochemical activity of the protein will be reduced or even damaged which is known with the term denaturation. Denaturation comes from the word "de" which means "out" and "natural". means “natural”. So denaturation is a departure from its original properties due to damage by various factors. The most fundamental damage to protein denaturation lies in its chemical structure, right? The primary structure is a peptide bond. As a result of damage to its chemical structure, proteins will loses its original physical and physiological characteristics. The occurrence of changes in protein physiology can eliminate properties natural properties such as enzymes and antibodies. Enzymes that experience denaturation will lose their biocatalytic properties and protein hormones will lose their regulatory function on the body's metabolism. Antibodies will loses its agglutination function against opposing antigens. Proteins that experience denaturation will eventually experience changes in physical properties such as: its molecular size, solubility, or consistency. Factors that can cause denaturation Proteins consist of chemical and physical factors. Chemical factors include the presence of disturbing chemicals protein charge, causing damage to protein chemical bonds. These factors can be acids, bases, inorganic salts, heavy metals,dehydrating agent(such as alcohol), urea, and organic solvents. Meanwhile, physical factors consist of temperature, UV light, pressure, mechanical factors such as shaking and etc. C. Experimental Principle a. Millon Test A positive reaction is indicated by the formation of a white precipitate which will turn into red when heated b. Biuret test The Biuret test is used to show the presence of peptide bonds in a polypeptide. A positive reaction is indicated by a marked change in color to blue, purple, or reddish in solution. D. Tools and Materials Tool Material 1. Dropper pipette 2. Measuring pipette 3. Test tube 4. Tube rack 5. Spirit lamp 1. Peptone 1% 2. 1% egg white solution 3. Millon's reagent 4. 2N NaOH solution 5. CuSO4 0.1N E. Work Procedures Millon Test a. Into each test tube, 3 ml of 1 type of protein and 5 drops of 1 type of protein were added Millon's reagent. Mix until homogeneous b. The solution is heated carefully, and observe the color change that occurs Biuret test 1. Put 2 ml of NaOH and 2 drops of CuSO4 into 2 clean test tubes, mix until homogeneous. 2. Into each tube, add 1 ml of 1 type of protein. mix until homogeneous. 3. Observe the color changes that occur. Ninhydrin Test 1. To 0.1 ml of 2% albumin solution (egg white) add 1 ml of 0.1N acetate buffer solution pH 5 and then add 20 drops of ninhydrin solution in acetone 2. Heat the mixture in an educational water bath for a few minutes, pay attention to the color that occurs Deposition with heavy metals 1. To 1 ml of albumin solution (egg white) add drop by drop 0.2% CuSO4 solution until a precipitate 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 again or is added to by adding excess reagent 2. Repeat each step (1) for solutions of 2% Pb-acetate, 2% HgCl2, 2% FeCl3, and 2% CuSO4 Protein Isoelectric Point 1. Into 5 test tubes each add 5 ml of 0.5% casein solution (milk). Next, to the five tubes, add acetate buffer pH 6.0; 5.3; 5.0; 4.1; and3,8 2. Shake the mixture well and note the degree of turbidity after: 0 minutes, 10 minutes and 30 minutes. 3. After 30 minutes, the five tubes above are heated in a boiling water bath for 30 minutes. The fastest formation of precipitate/turbidity occurs near the isoelectric point of the protein solution. Pay attention to what happens and note it down.