05 Enzymes.pdf

ENZYMES Department Of Molecular Medicine School Of Medicine And Dentistry KNUST  OBJECTIVES To determine the achromic point of an enzyme To study the effect of temperature on the activity of salivary amylase. Good morning  WHAT ARE ENZYMES...

ENZYMES Department Of Molecular Medicine School Of Medicine And Dentistry KNUST  OBJECTIVES To determine the achromic point of an enzyme To study the effect of temperature on the activity of salivary amylase. Good morning  WHAT ARE ENZYMES? Biological catalysts mostly made up of proteins. Ribozymes are catalytic nucleic acids They mediate a lot of bodily reactions Usually work by lowering the activation energy levels. Good morning  CLASSIFICATION OF ENZYMES Based on Composition Based on Mechanism Simple enzyme of action Complex enzyme Oxido-reductases Transferases Hydrolases Lyases Isomerases Ligases Good morning  ROLES OF ENZYMES Enzymes function in the following processes: Digestion Respiration Metabolic reactions Muscle and nerve function DNA reactions Good morning  DIAGNOSTIC ENZYMES Enzymes used for diagnosis or prognosis. Enzymes are preferred in diagnosis of some disease states, because of their substrate specificity their activity can be quantified even in the presence of other proteins. Good morning  DIAGNOSTIC ENZYMES Disease states usually lead to moderate or extensive tissue damage depending on the time of onset and severity of the disease. Such conditions are usually associated with the release of enzymes specific to the diseased organ or tissue into circulation and this results in an increase in activity of such enzymes in body fluids. Good morning  CLINICAL RELEVANCE OF ENZYMES Biomarkers: They are the preferred markers in various disease states. E.g. Creatine kinase for muscle weakness and injury. Therapeutic accelerators: Enzymes are typically used as medicines to salvage enzyme deficiencies in patients. E.g. Papain is administered orally after food for easier digestion in geriatric patients Disinfection: Enzymatic detergents designed specifically for cleaning reusable medical devices have been around for decades. Proteases are used to clean wounds and thus accelerate the healing method. Analytical tests (Biosensors) : Diabetics use strips of paper fertilized with aldohexose enzyme to observe their glucose. Eg Glucose oxidase (GOD) is a widely used biosensor. Good morning  FACTORS AFFECTING ENZYME ACTIVITY Temperature Substrate concentration pH Enzyme concentration Cofactors Product concentration Good morning DEMONSTRATION OF FACTORS THAT AFFECT ENZYME ACTIVITY USING PTYALIN Good morning SALIVARY AMYLASE (PTYALIN) Salivary α-amylase breaks starch down into maltose and dextrin. It breaks large, insoluble starch molecules into soluble starches (amylodextrin, erythrodextrin, achrodextrin) producing successively smaller starches and ultimately maltose. Good morning  PROPERTIES OF SALIVARY AMYLASE Protein in nature Optimum Temp: 37 °C Optimum pH: 6.8 Activators : halogen ions especially Cl− Duration of action: 15 minutes Good morning  STARCH Amylose is a linear chain of glucose units that are linked by α-1,4 bonds (20-30%) Good morning  STARCH Amylopectin is a branched molecule in which glucose units are linked by α-1,4 bonds in the straight chain region in addition to α-1,6 bonds at the branching points (70-80%). Good morning Salivary amylase hydrolyses only the α-1,4 bonds that link glucose units. Therefore, starch and glycogen which consist of α-1, 4 bonds, are hydrolysed by amylase while cellulose, which consists of β-1,4 bonds, is not hydrolysed by salivary amylase Good morning  DETERMINATION OF ACHROMIC POINT Achromic point is defined as the time taken by salivary amylase to hydrolyse starch to achrodextrin at optimum conditions Good morning  PROCEDURE ONE 1. Prepare water bath at 37°C 2. Add 2 drops of iodine each into the wells of the microtitre plate 3. Select 3 wells for Controls. Into one, put a drop of starch, then put a drop of amylase enzyme, into another, and leave one with iodine only. 4. Label one test tube “Ach + group number” and add 2ml starch solution to 2ml amylase solution. Mix well and place the tube solution immediately in the water bath. Good morning 4. Start a Stopwatch, at one minute intervals take a drop of the enzyme-starch mixture and add it to the iodine in one of the wells (except controls), the colour becomes blue-black. 5. Record the time taken for the colour to change from blue to the iodine colour. This represents achromic point. Good morning  FORMAT OF RESULTS TIME OBSERVATION INFERENCE Starch 1 minute Colour Change/ Present (10 mins) Achromic Time What has happened to the starch? Explain this observation…….. Good morning  EFFECT OF TEMPERATURE ON THE ACTIVITY OF SALIVARY AMYLASE Good morning  PROCEDURE TWO 1. Label the remaining 5 test tubes “37°C ”, “100°C. M”, “100°C. T”, “0°C. M” & “0°C. T ”. 2. Put 1 ml of starch solution in each of the test tubes and 1ml of enzyme solution. 3. Immediately put test tubes “37°C ” in a 37°C bath, test tube “0 °C. M” and ’’0°C. T” in ice bath. and test tube “100°. M and 100 °C. T” in a boiling water bath. 4. Let the test tubes sit until the achromic time. Good morning  PROCEDURE TWO 5. The remaining 5 test tubes should be labeled "37°C", "100°C. M", "100°C. T", "0°C. M", and "0°C. T". 6. Add 1ml of the enzyme solution and 1ml of the starch solution to each test tube. 7. Place test tubes labeled "37°C", and "0°C. M" and "0°C. T" in baths of 37°C and ice, respectively, right away. Put a test tube labeled "100°. M and 100 °C. T" in a pot of boiling water. 8. Let the test tubes sit until the achromic time. Good morning  PROCEDURE TWO 9. After the achromic time, add 2 drops of iodine to each of the test tubes labeled "37 °C," "0 °C. M," and "100 °C. M." 10. Depending on the byproducts of starch digestion in each of the three test tubes, it will be discovered that iodine produces a variety of colors. 11. Describe the ensuing observations 12. Bring the test tubes labeled "0 °C. T" and "100 °C. T" back to 37 °C and let them sit there for ACHROMIC TIME 13. Add 3 to 5 drops of iodine. Track any color changes. Good morning The expected colours may be as follows: Tube1 (37 °C. ): Brown (achrodextrinis the product of starch digestion , salivary amylase is active and 37°C is optimum for its maximal activity. Tube 2 (0 °C. M): Violet (erythrodextrin or amylodextrin) may be the product of starch digestion which is incomplete by the end of achromic point time. Room temp. is not optimum for the enzyme activity Tube 3(100 °C. M): Blue , starch is not digested, amylase is inactive and boiling, denatures the enzyme Good morning  Tube 4 (0 °C. T): Brown (achrodextrinis the product of starch digestion , salivary amylase reactivates at 37°C as it is the optimum for its maximal activity. Tube 5 (100 °C. T) : Blue , starch is not digested, amylase is inactive and boiling, denatures the enzyme Good morning  FORMAT OF RESULTS TEST OBSERVATION INFERENCE 1 ml STARCH + 1 ml Enzyme at Colour Changes / State inferences set temperature Colours formed and give for achromic reasons for such time observations THANK YOU Good morning