Teaching Kit Manual Cat No. New Cat No. KT89 106209 Revision No.: 00140806
CONTENTS Page No. Objective 3 Principle 3 Kit Description 4 Materials Provided 5 Procedure 6 Result 12 Interpretation 17 ORDERING INFORMATION 19 1
Objectives: The kit is designed for students to learn the principle of enzyme kinetics. Principle: Enzymes are the reaction catalysts of biological systems. They have high degree of specificity for their substrate. Enzymes function in aqueous solutions under very optimum / mild conditions of temperature and ph. The study of reactions rates and the influence of changes in environmental factors (such as temperature, ph etc) is known as Kinetics. Enzyme kinetics is that branch of enzymology which deals with factors affecting rates of enzyme - catalyzed reactions. The most important factors are as follows: (1) Enzyme concentration (2) Substrate concentration (3) Temperature and (4) ph 2 3
Kit Description: Using this kit, students will study the effect of enzyme peroxidase on the substrates (guaiacol and hydrozen peroxide). reacts with H 2 O 2 in the presence of peroxidase to form guaiacol oxide, which is a brown colored product. The speicific reaction is () 4H- R-O-H + 4 H 2 O 2 DOR-O-O-R + 8 H2O (GUAIACOL) R-O-O-R In addition to carrying out the reaction, students will study the effect of enzyme, substrate, ph and temperature on the rate of the enzyme catalysed reaction. KT89 : The kit is designed to carry out 5 experiments. Duration of experiment: Approximately 3 hours Materials Provided: The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival. Materials Quantity KT89 (5 expts.) Store (enzyme) 1 mg -20 C 15 ml -20 C H 2 O 2 1 ml 4 C Assay buffer (ph 3.0) 30 ml 4 C Assay buffer (ph 5.0) 30 ml 4 C Assay buffer (ph 7.0) 30 ml 4 C Assay buffer (ph 9.0) 30 ml 4 C Materials Required: Equipment : Spectrophotometer. Glassware : Test tubes. Other Requirements: Micropipette, Tips, 4 5
Note: Reconstitute the provided with 1 ml of distilled water before use. Store at 4 C and use within 6 months. Just before each experiment prepare working concentration of solution from 1mg/ml stock as follows. Prepare 10 mm solution by adding 60 µl to 24 ml distilled water. Stable for 1day. Prepare the working concentration of hydrogen peroxide just before starting each experiment. (170 µl to be made up to 5 ml with distilled water for one experiment). Procedure: Step 1: Standard/Base line test 1. Add the reagents in tubes as given in table 1. Water (ml) 1. 80 800 ---- 3.12 2. 80 ---- 80 3.04 2. Adjust the reading to zero absorbance with tube 1 blank and start the reaction by adding 800ul of solution to tube 2 and read the absorbance of the mixture at 470 nm at every 30 seconds interval for 3 minutes. 3. Enter the reading as shown in the result. I. Effect of Enzyme concentration: 1. Perform two tests, one with double the amount of enzyme and another with half the amount of enzyme. 2. Prepare blanks separately for tests A and B since it contains double the amount and half the amount of enzyme respectively. A. Double the amount of enzyme (2X) 1. Add the reagents in tubes as shown in table 2 Water (ml) 1. 80 1600 ---- 2.32 2. 80 ---- 80 2.24 2. Adjust the reading to zero absorbance with tube 1 blank and start the reaction by adding 1600 µl of solution to tube 2 and read the absorbance of the mixture at 470 nm at every 30 seconds interval for 3 minutes. 3. Enter the reading as shown in the result. B. Half the amount of enzyme (0.5X) 1. Add the reagents in tubes as shown in table 3. Water (ml) 1. 80 400 ---- 3.52 2. 80 ---- 80 3.44 6 7
II. Effect of Temperature: 1. Perform five tests, at different temperatures viz., 10 C, RT, 37 C, 55 C and 70 C. 2. Add the reagents in tubes as shown in table 4. 3. Adjust the spectrophotometer to zero absorbance with tube 1 and incubate the remaining tubes at the respective temperature as mentioned in the table for 10-15 min. 4. Start the reaction by adding 800 µl of solution to each tubes at a time and quickly read the absorbance of the mixture at 470 nm at every 30 sec interval for 3 min. 5. Enter the reading as shown in the result table 4. Temperature H 2O 2 Water (ml) Blank 1. 80 800 ------ 3.12 10 C 2. 80 ------ 80 3.04 R.T 3. 80 ------ 80 3.04 37 C 4. 80 ------ 80 3.04 55 C 5. 80 ------ 80 3.04 70 C 6. 80 ------ 80 3.04 III. Effect of substrate: 1. Perform two tests, one with double the amount of substrate and another with 10 fold increase in the amount of substrate. 2. Add the reagents in tubes as shown in table 5. 3. Adjust the spectrophotometer to zero absorbance with tube 1 and start the reaction by adding 800 µl of solution to each tubes at a time and quickly read the absorbance of the mixture at 470 nm at every 30 sec interval for 3 min. 4. Enter the reading as shown in the result. Amount of substrate Water (ml) ------ 1. 80 800 ---- 3.12 2 fold (2X) 2. 80 ---- 160 2.96 10 fold (10X) 3. 80 ---- 800 2.32 8 9
IV. Effect of ph: 1. Perform four tests by varying the ph of the buffer viz. 3.0, 5.0, 7.0, 9.0. 2. Prepare different blanks for different sets of ph experiments. 3. Adjust the reading to zero absorbance with tube 1 blank and start the reaction by adding 800 µl of solution to tube 2 in each experiment. A. ph 3.0 Add the reagents in tubes as shown in table 6 Assay buffer (ph 3.0) (ml) 1. 80 800 ---- 3.12 2. 80 ---- 80 3.04 C. ph 7.0 Add the reagents in tubes as shown in table 8. Assay buffer (ph 7.0) (ml) 1. 80 800 ---- 3.12 2. 80 ---- 80 3.04 D. ph 9.0 Add the reagents in tubes as shown in table 9. Assay buffer (ph 9.0) (ml) 1. 80 800 ---- 3.12 2. 80 ---- 80 3.04 B. ph 5.0 Add the reagents in tubes as shown in table 7. Assay buffer (ph 5.0) (ml) 1. 80 800 ---- 3.12 2. 80 ---- 80 3.04 10 11
Result: Tabulate the readings (A-470) and plot the graphs as follows: a. Baseline Run (1X) Time in seconds (X-axis) Absorbance (Y-axis) 0 0.075 30 0.173 60 0.290 90 0.442 120 0.589 150 0.703 180 0.814 B. Effect of Enzyme Concentration Time in seconds (X-axis) A470 with different conc. 2X 0.5X baseline baseline 0 0.100 0.078 30 0.190 0.198 60 0.755 0.303 90 1.100 0.403 120 1.333 0.515 150 1.639 0.597 180 1.830 0.687 Effect of Enzyme Concentration 2 A_470 1.5 1 0.5 0.5X 1X 2X 0 0 50 100 150 200 Time(sec) 12 13
C. Effect of Temperature Time A-470 at different Temperature in Sec 10 C RT 37 C 55 C 70 C 0 0.012 0.076 0.049 0.014 0.005 30 0.024 0.173 0.160 0.043 0.033 60 0.039 0.290 0.246 0.095 0.053 90 0.059 0.442 0.334 0.134 0.073 120 0.068 0.589 0.421 0.174 0.093 150 0.081 0.703 0.514 0.198 0.111 180 0.097 0.814 0.619 0.214 0.127 D. Effect of ph Time A-470 a different ph in Sec 3.0 5.0 7.0 9.0 0 0.022 0.022 0.003 30 0.083 0.084 0.013 60 No Colour 0.135 0.148 0.022 90 Development 0.196 0.202 0.032 120 0.259 0.296 0.039 150 0.322 0.383 0.047 180 0.385 0.454 0.054 Effect of temperature 10 RT 37 Effect of ph 0.9 0.8 0.7 55 70 0.5 A_470 0.6 0.5 0.4 0.3 0.2 0.1 0 0 50 100 150 200 Time(sec) A_470 0.4 0.3 0.2 0.1 3 5 7 9 0 0 50 100 150 200 Time(sec) 14 15
E. Effect of Substrates Time in A-470 a different Substrate Concentration Sec 2X Baseline 10X Baseline 0 0.026 0.048 30 0.086 0.170 60 0.148 0.283 90 0.209 0.411 120 0.279 0.548 150 0.346 0.675 180 0.406 0.790 Interpretation: a) When substrate concentration is held constant and the amount of enzyme varied, a linear increase or decrease in the activity rate was observed. In the experiment of the reverse type, a non-linear hyperbolic relationship between activity and substrate concentration were observed. b) The rate of enzyme catalysed reaction increases with temperature. The reaction rate approximately double itself for every 10ºC to 15ºC rise in temperature as shown in graph. However, the thermal denaturation sets in as the temperature is raised. Therefore, as the temperature is raised, the rate passes through a maximum and then falls. Effect ofsubstrate concentration A_470 1 0.8 0.6 0.4 0.2 0 0 50 100 150 200 Time(Sec) 10X 1X 2X Rate of Reaction 16 17
c) Enzyme activity is ph dependent. It is found from the experiment, over a limited range of ph and within this range, the ph activity curve is roughly bell shaped and shows a maximum at a point known as the optimal ph. Most enzymes have ph optimum at region 6-8. At extreme ph values protein denaturation occurs with a loss of enzyme activity. Ordering Information Product Size Cat # 1 Pack KT89 Teaching Kit (Consumables for 5 experiments) Activity Email: Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com 18 19
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