EXP 4 - Enzyme Activity - Biology Laboratory - PDF

Sara Khasawneh Experiment - 4 BioLab Sara Khasawneh Experiment - 4 BioLab Experiment 4 : Enzyme Activity  What is an enzyme? - Definition : A biological catalyst that speed up the rate...

Sara Khasawneh Experiment - 4 BioLab Sara Khasawneh Experiment - 4 BioLab Experiment 4 : Enzyme Activity  What is an enzyme? - Definition : A biological catalyst that speed up the rate of chemical reactions in living organisms by lowering the activation energy without being changed or used up. ‫هو عبارة عن محفز حيوي للتفاعالت الكيميائية التي تزيد من سرعة التفاعل عن طريق تقليل طاقة المعقد المنشط‬.‫دون أن تتغير أو تستهلك أثناء التفاعل‬ - Notes about Enzymes: 1. Most of them are proteins with 3D (Globular) structure. 2. Enzymes are specific and speed only one type of reaction..‫أغلب االنزيمات تعتبر بروتينات كروية الشكل ولكل إنزيم تفاعل محدد يزيد من سرعته‬ - Nomenclature of Enzymes:‫تسمية االنزيمات‬ Enzymes are often named by adding “-ase” Example: Dehydrogenase: An enzyme that remove hydrogen.  How do enzymes catalyze reactions? ‫كيف‬ ‫يحفز االنزيم التفاعالت‬ - By lowering the activation energy required to: 1. Break bonds in reactants 2. Form new bonds in products - The activation energy is the least possible amount of energy required to start a reaction ‫ويتم ذلك عن طريق تقليل الطاقة الالزمة لبدء التفاعل طاقة التنشيط واذا‬ ‫وصلنا لهي الطاقة بتتكسر الروابط بالمتفاعالت وتبنى روابط جديدة‬  How do enzymes work? ‫كيف تعمل االنزيمات‬ - By binding to a substrate (reactant) at its active site to form an enzyme-substrate complex Sara Khasawneh Experiment - 4 BioLab - By converting reactants to products without being changed, meaning that they can be used repeatedly ‫ترتبط بالمتفاعالت وتكون معقد مكون من االنزيم والمتفاعالت ثم يحولها لنواتج من دون أن يتغير او يستهلك أثناء التفاعل‬.‫لذلك يمكن استخدامه أكثر من مرة‬  What is the enzyme-substrate complex? ‫متفاعالت‬-‫ما هو معقد انزيم‬ - The intermediate that is formed when a substrate binds to an enzyme its active site. - The active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction ‫ وهو المكان الذي ترتبط فيه المتفاعالت‬.‫هي حالة وسطية تحدث لحظة ارتباط المتفاعالت بالموقع النشط لالنزيم‬.‫ليحدث التفاعل الكيميائي‬  What is meant by “substrate-specific”? ‫ماذا نعني بقولنا محدد للمتفاعالت‬ - Enzymes are substrate-specific, meaning that they catalyze specific reactions ‫يعني إنه هاد االنزيم محدد فقط لعمل تفاعل معين بس ما بيقدر يعمل التاني‬. - LIKE: 1. Proteases break peptide bonds in proteins 2. Amylases catalyze the hydrolysis of starch 3. Catalase: breaks down H2O2  What is catalase? ‫ما هو الكاتاليز‬ - It is an enzyme found in most living organisms - In lab, the source of catalase will be chopped potatoes - In human, liver produced catalase. Sara Khasawneh Experiment - 4 BioLab - What is the substrate of the enzyme catalase? Hydrogen peroxide (H2O2) 2H2O2 + catalase → 2H2O + O2 ‫هو انزيم موجود في أغلب الكائنات الحية للتخلص من بيروكسيدالهيدروجين (فوق أكسيد الهيدوجين) وهي مادة سامة للكائنات‬ ‫الحية فبحللها وبنتج منها ماء وأكسجين الي حنستخدمه بالالب مصدره عصارة البطاطا إلنه منطقيا ما بقدر أجيب كبد بني آدم‬.‫وآخد منه هاالنزيم‬  What variables affect the function of an enzyme? ‫ما هي العوامل المؤثرة في‬ ‫عمل االنزيم‬ 1. Enzyme concentration: ‫تركيز االنزيم‬ - ↑[enzyme] = ↑rate (to a certain extent) ‫كل ما زاد تركيز االنزيم بزيد سرعة التفاعل وهي منطقيا إنه االنزيم بضل يشتغل طول ما في متفاعالت‬ ----------------------------------------------------------------------------------------------------------- - What if the substrate has a certain limit? ✓ At the point of saturation, all substrates are bound. ✓ This means that: Adding more enzymes will not increase the rate of reaction. ✓ The substrate becomes the limiting factor. ✓ Not all enzyme molecules can find substrate Sara Khasawneh Experiment - 4 BioLab Practical Part: 1. Obtain 6 test tubes and a test tube rack (Holder) per group. ‫بنجيب ست أنابيب اختبار وبنحطهم بالقاعدة الي بتمسك األنابيب‬ 2. Add certain drops of H202 to each tube. ‫بعدها بنحط عدد معين من بيروكسيد الهيدروجين‬ 3. Add certain drops of H2O and no potato extract (no enzyme) to tube #1. ‫بنضيف كمية معينة من المي عاالنبوب األول من دون ما أضيف الكاتاليز أو عصارة البطاطا‬ 4. Using a dropper repeat step3 to the remaining 5 tubes increasing by three drops the amount of potato extract (here we increase the enzyme)..‫ قطرات‬3 ‫بنضيف عاألنابيب الي ضلت كاتاليز وبكل أنبوب بنزيد عدد القطرات‬ 5. after timing for 60 seconds, mark the maximum height of bubble column(foam) by a marker..‫بعد ما ضفنا عليهم بنستنى دقيقة وبنحدد ارتفاع الرغوة في كل أنبوب‬ - The result: you will notice that by increasing the amount enzyme (catalase/potato extract) the enzymatic activity will increase. 2. Substrate concentration: ‫تركيز المواد المتفاعلة‬ - ↑[substrate] = ↑rate (to a certain extent( ‫كل ما زاد تركيز المواد المتفاعلة زاد معدل التفاعل لحد ما يضل فيه عندنا انزيمات قادرة على االرتباط‬ ‫بمتفاعالت وهون بيثبت النشاط‬ - At the point of saturation, all substrate are bound to enzymes. - This means that: Adding more substrate will not increase the rate of reaction. - The enzyme becomes the limiting factor. - Not all substrate molecules can find enzyme to bind. Sara Khasawneh Experiment - 4 BioLab Practical Part: 1. Obtain 8 test tubes and a test tube rack (Holder) per group. ‫ أنابيب اختبار وبنحطهم بالقاعدة الي بتمسك األنابيب‬8 ‫بنجيب‬ 2. add a certain drops (using dropper) of potato extract to each tube..‫بعدها بنحط عدد معين من الكتاليز الموجود بعصارة البطاطا‬ 3. add a certain drops of water (H2O) and no H2O2 to tube #1. ‫بنضيف كمية معينة من الماء على االنبوب األول بدون ما أضيف بيروكسيد الهيدروجين‬ 4. 4-using a dropper repeat step 3 to the remaining test tubes increasing by three drops the amount of H2O2.(here we increase the substrate ) ‫ قطرات‬3 ‫بنضيف عاألنابيب الي ضلت بيروكسيد الهسيدروجين وبكل أنبوب بنزيد عدد القطرات‬ 5. after timing for 60 seconds, mark the maximum height of bubble column(foam) by a marker..‫بعد ما ضفنا عليهم بنستنى دقيقة وبنحدد ارتفاع الرغوة في كل أنبوب‬ - The result: you will notice that by increasing the amount of substrate (H2O2) the enzymatic activity will increase until certain limit = 20 drops of H2O2 DO NOT STOP….! Sara Khasawneh Experiment - 4 BioLab 3. Temperature: - ↑Temperature= ↑rate to the OPTEIMUM then ↓rate ‫كل ما زدنا درجة الحرارة سرعة التفاعل بتزيد لتوصل درجة الحرار المثلىبس بعدين درجات الحرارة العالية‬. ‫بتدمر االنزيم فسرعة التفاعل بتقل‬ - Effects of temperature on enzyme activity Optimum T: ✓ Molecules accept their highest kinetic energy. ✓ Then the greatest number of molecular collisions take place. ✓ Shows the highest enzyme’s activity. ✓ For Human enzymes the range of optimum temperatureis 35o – 40oC Heat: Increase beyond optimum T: ✓ Disrupts the weak bonds ( H and Ionic bond ): 1. in enzyme 2. between enzyme and substrate ✓ Denature the enzymes = Lose the 3D shape(3o structure) Cold temperature: Decrease away from optimum T: ✓ Lower the kinetic energy of molecules ✓ Molecules move slower ✓ Decrease the collision between enzyme and substrate ✓ At extreme low T the enzyme will be inactivated and this will inhibit the reaction Practical Part: 1. Obtain 10 test tubes and a test tube rack (Holder) per group. 2. using a pipette, measure 1ml of H2O2 into 5 of the 10 tubes. 3. using a dropper, add certain drops of potato extract to the other 5 tubes. 4. place one test tube of each the H2O2 and the catalase into each of ice pocket, refrigerator, room temperature, water bath(50C'), boiling bath. 5. leave the tubes for 10 minutes. 6. then pour the H2O2 into the catalase, which have been incubated in the same condition. Sara Khasawneh Experiment - 4 BioLab - The result: will notice that its highest at the optimum temperature which are the tubes which were placed at room temperature = 25 4. pH: ‫درجة الحموضة‬ - As pH moves away from optimum = ↓rate - Optimum pH: It is the pH at which the enzyme is most effective - For most enzymes, it lies in the range from pH 5 to pH 9 - A change in pH can: ✓ Change the charges of Carboxyl group and Amino group of the amino acids. ✓ Alter the ionization of the R groups of amino acid. ✓ Affect the hydrogen bond and covalent bond between amino acid of the active site and the substrate. ✓ These changes can affect hydrogen bonding and attraction within the protein and between the protein and other molecules. ✓ Extremely high or low pH affect the 2o and 3o structure of the enzyme, and results in a complete loss activity for most enzymes (Denaturation of enzyme). Practical Part: 1. Obtain 3 test tubes and a test tube rack (Holder) per group. 2. using a pipette, add certain drops of potato extract to each of the three tubes. 3. using a dropper, add certain drops of lemon juice, H2O, and NaOH to test tubes 1,2,3 respectively. 4. after 10 minutes, add certain drops of H2O2 to each tube. 5. after 60 seconds measure the height of the foam, Sara Khasawneh Experiment - 4 BioLab - The result: you will notice that its highest at the optimum PH =7 which is the tube filled with water in our experiment.  Some notes: 1. Why does an increase in temperature lead to an increased rate of reaction at first? - Temperature is a measure of the speed at which molecules are moving - ↑Temperature = ↑Speed of molecules 2. If you increase the temperature to extremes the enzyme will be desaturated ‫ما بيقدر‬ ‫ يرجع لوظيفته‬But when it decrease to the extreme it will be just in active. 3. We have other variables affect the function of an enzyme like Cofactors, Salt concentration, Pressure and Surface area. 4. Optimum pH It depends on the type of the enzyme and on where it is normally acts ( Localized condition ) Pepsin (stomach) > pH = 2-3 Trypsin (Intestine) > pH = 8 5. Hydrogen peroxide is a toxic chemical which can burn skin 6. Immediately rinse any spills with water 7. Wash hands before leaving the lab 8. To prepare catalase enzyme: we cut 500g potato and we add them with 500 ml of water in blinder for 5-10 mins then filter the m mixture with some layers of cheese cloth held with a funnel to make a stock of catalase We close the bottle immediately and tightly because it contains catechol which converts in presence of oxygen by catecholase to benzoquinone which affect the catalase activity. We keep it in ice bucket till we use it to lower the enzymes activities.