Properties of Enzymes and Competitive Inhibitors

Index Page Abstract. . . 3 demonstration. . .. 3 Materials and Chemicals used.. .. . .. 3 Procedures.. 4 T fitteds 5-7 Results.. 8 Discussion. .. 8 closing. . 8 Works Cited .. 9 Properties of Enzymes and Competitive Inhibitors. Abstract Properties of enzymes were found in this experimentation and some other factors, which affect enzyme activity.Enzymes are catalyst they catalyse very specific replys. Results relating to the active site of specific enzymes played a big role while perfor arcminuteg this experiment. The purpose of this experiment was to equip how inhibitors affect enzymes activity by competing for the active site once against substrates. Introduction Cells have the ability to perform chemical reactions that at normal temperature outside the body proceed too slowly to support life. Cells are able to perform some reactions rapidly because they possess protein catalyst called enzymes. Enzymes are proteins that catalyze (i. . , increase the rates of) chemical reactions. Each enzyme has a unique spherical shape, a small portion of which functions as an active site satisfactory of binding to specific reactants or substrates. It was hypothesized that enzyme dumbness, temperature, and inhibitors will affect the properties and abilities of the enzyme. Materials 1Wax Marking Pens one hundred fifty ml Beakers 3 400 ml Beaker 1 container of parafilm 1 station of 20 spec tubings 1 regular experiment provide stand 1 small turn up thermionic vacuum tube rack 1 street corner Kimwipes Eye Droppers 1 thermometer 2-10ml Graduated Cylinders 1 Spectrophotometer 7 C waterbath with test tube racks Solutions 1 flasks of pH 7 buffered ONPG 1 flask of milk sugar 8% 1 flask of pH 7 buffered 1 flasks of 8% beta galactosidase Procedure 1. Obtain five test tubes and label them (i. e. A, B, C, D, E) 2. victimization a 10 ml graduated cylinder trust agate line It is very primary(prenominal) to add enzyme last. 1 ml of pH 7 buffered ONPG + No Lacto se 8%(0ml) +(1 ml pH buffer) + Enzyme (1ml) solutions into tube A. 0% Lactose. 3. Using a 10 ml graduated cylinder put 1 ml of pH 7 buffered ONPG + Lactose 8% (. 25ml) +(. 75ml pH buffer) + Enzyme (1ml) solutions into tube B. % Lactose. 4. Using a 10 ml graduated cylinder put 1 ml of pH 7 buffered ONPG + Lactose 8% (. 5ml) +(. 5ml pH buffer) + Enzyme (1ml) solutions into tube C. 4% Lactose. 5. Using a 10 ml graduated cylinder put 1 ml of pH 7 buffered ONPG + Lactose 8% (. 75ml) +(. 25ml pH buffer) + Enzyme (1ml) solutions into tube D. 6% Lactose. 6. Using a 10 ml graduated cylinder put 1 ml of pH 7 buffered ONPG + Lactose 8% (1ml) +(0ml pH buffer) + Enzyme (1ml) solutions into tube E. 8% Lactose. 7. Cover each of the tubes with parafilm and place the tubes in the 37 C waterbath for 30 transactions. . After 30 minutes, determine if the reaction has occurred in each tube, and scar motley in color. 9. try on tube E acted as our swan test tube because no matched inhibitor was adde d. Lactose was the competitive inhibitor for this reaction into the test tube. Note Because the result on steps 4 and 6 were not accurate for our particular experiment, steps 4 and 6 were performed twice. The avocation turn off and graph express the results after the measurements and mixing. Table 1. Measurements after mixing the solutions into the test tubes.Solutions pH 7 Buffered ONPG (ml) Lactose 8% (ml) pH buffer (ml) Enzyme B-Gal (ml) Total amount of mls. Test tube A 1 0 1 1 3 Test tube B 1 0. 25 0. 75 1 3 Test tube C 1 0. 5 0. 5 1 3 Test tube D 1 0. 75 0. 25 1 3 Test tube E 1 1 0 1 3 This evade represents the total amounts of each solution added to each test tube in severalize to get 3 mls for each test tube. This table is used only to represent how the result will look like. interpret 1. Measurements after mixing the solutions into the test tubes. This graph depicts the contents inside the test tubes after mixing the mentioned solutions.Measurement of O-nitrophenol. ( ONPG) Although the appearance of jaundiced in the tubes indicated that O-nitrophenol was present, the color, alone, did not prescribe us how much was present. It was possible to measure the amount of O-nitrophenol present by measuring the intensity of the yellow with a spectrophotometer. 1. The contents of the 5 tubes were poured into spec 20 tubes. The positions were labeled, but the spec tubes were left clear in run to have an accurate measurement absorbance. 2. Test tube E acted as the stop tube for this, since that tube did not contain inhibitor.Note Absorbance 420nm in this experiment will be a measure of the concentration of the O-nitrophenol molecules in each of the solutions. Using the Spectrophotometer The spectrophotometer was an instrument intentional to measure the amount of light familial through solutions, or absorbed by substances in the solution. Light of a specific wavelength is emitted from a special bulb and passed through a tube containing a substance soluti on. The greater concentration of those particles the greater the absorbance. It is very important to select the most appropriate wavelength of light for use.These procedures were followed in order to company up the Spectrophotometer. 1. 420 nm was the wavelength to use in the inhibitor experiment lab designed because O-nitrophenol maximally absorbs a light at 420. 2. The Spectrophotometer was zeroed out with the dominate pommel so that the needle reads 0% transmittance on the upper scale. 3. The check out tube A was put in the holder, and the lid was closed. The light control knob was adjusted so that the needle could read 100% transmittance. 4. The control tube was removed from the holder. The lid was then closed noticing the needle again read 0% transmittance. 5.All other test tubes were placed into the Spectrophotometer and read as well. 6. Data for these results was recorded on the following table. Table 2. Effect of competitive inhibitor concentration lactose on the product ion of O-nitrophenol. Effect of Competitive Inhibitor constriction on production of ONGP Product Tube Inhibitor Concentration Intensity of yellow Absorbance ? moles of ONPG produced/30min ? moles of ONPG produced/min A 0% ++++ 1. 55 38. 75 1. 291666667 B 2% +++ 0. 43 107. 5 3. 583333333 C 4% ++ 0. 13 32. 5 1. 083333333 D 6% + 0. 02 5 0. 166666667 E 8% 0 0 0 0Calculation of ? moles O-nitrophenol produced per minutes. Ex. Tube A ? moles of ONPG produced/30min Absorbance/0. 004= ? moles of ONPG produced per 30min 0. 155 / 0. 004= 38. 75 ? moles Ex 2 Tube A ? moles of ONPG produced/min ?moles of ONPG produced per 30min/ 30min 38. 75 /30=1. 291666667 ? moles of ONPG produced/min From the absorbance data that was measured the O-nitrophenol produced per minute was calculated. 1. Each ? mole of O-nitrophenol produced an absorbance of 0. 004. The absorbance measured was divided by 0. 004 to determine the number of ? moles produced during the experiment.The values were recorded in table 2 , fifth column. 2. The measurements that were obtained in the fifth column were divided by 30(number of minutes left in the waterbath) to obtain the number of ? moles of O-nitrophenol produced per minute. Graph 2. Absorbance measurements for inhibitor concentration lactose on the production of O-nitrophenol. Absorbance Absorbance Test Tubes Test Tubes Results According to the hypothesis that temperature, enzyme concentration, and concentration will affect the properties and functions of the enzymes. The hypothesis was supported because graph and tables express the change in absorbance, and ? oles produced. Discussion The tables were able to depict the result in order to get better and accurate results for this particular experiment. Measurements have to be performed with precaution, qualification sure the enzyme and the contents are mixed properly and at the aforementioned(prenominal) time. Conclusion Enzyme activity can be affected by other molecules. Inhibitors are molecules tha t decrease enzyme activity activators are molecules that increase activity. Activity is in addition affected by temperature, chemical environment, change in pH, and the concentration of substrate.