The Effect Effects of Temp & PH on Enzymes - Lab Report Example

College: The effect effects of temp & PH on Enzymes Introduction Enzymes are special types of proteins that act asbiocatalysts in various biological processes. Without enzymes, most chemical reactions that maintain a viable organism would not occur. One main characteristic of enzymes is that they are very selective because they catalyze only one particular reaction. This is because they have unique active sites into which only specific substrates can fit. Enzymes bind temporarily to one or more of the reactants of the reaction they catalyze and in doing so; they lower the amount of activation energy needed and thus speed up the reaction (Groves, 1997). The activity of enzymes is highly affected by changes in pH and temperature and as such each enzyme works best at a given pH and temperature (Jencks, 1987). Changes in pH alter the state of ionization of charged amino acids that may play a crucial role in substrate binding and/or the catalytic action itself. Similarly, hydrogen bonds are easily disrupted by increasing temperature which disrupts the shape of the enzyme such that its affinity for its substrate diminishes (Groves, 1997). Objectives The purpose of this paper is to investigate the effects of pH and temperature on enzyme reaction. The paper will also analyze the results of the experiment. Materials & Methods In this experiment, each group of four students was provided with a tube of concentrated -amylase that was labeled A, B or C. The tube with enzyme was kept on ice. Each group performed part 1 and 2 of the experiment. Part 1: The effect of temperature on -amylase Activity. First, -amylase preparation C was recorded and then one test tube was labeled "blank" and five others as 40C, 230C, 370C, 650C and 1000C. 1ml of 1% starch solution at pH 7 was added to each test tube, whereby the starch was the substrate for the reaction. Each tube was placed in a water bath that was set as one of the indicated temperatures. The blank and the 230C were placed at room temperatures while the 40C on ice. All the tubes were allowed to equilibrate to the desired temperatures for ten minutes. A fresh dilution of the unknown -amylase was made by mixing 100l of the concentrated enzyme stock with 9.9 of dH20 shaking to mix. The stock and the diluted solutions were kept on ice. All the tubes were retrieved after 10 minutes of pre- incubation step. 1ml of dH20 was added to the blank tube only. To the other five tubes, a timer was set at 12 min. and the count down began immediately the moment 1ml of dilute -amylase was added to each of the five tubes, shaking to mix after which each tube was returned to its appropriate temperature. In order to test maltose, 1 ml of maltose color reagent was added to each tube including the blank. The tubes were vortexed after which they were placed in a boiling water bath for exactly 15 mins. Timing was started immediately the maltose color reagent was added to the first tube. The maltose color reagent denatures all of the enzymes hence stopping any enzymatic reactions. The tubes were placed on ice until they cooled to room temperature after which 9 ml of dH20 was added to each tube shaking to mix. Using the blank to zero the spectrophotometer A540nm was measured of all samples and the results were as in the table 1 and Table 2 below. Part 2: The effect of pH on -amylase activity First, -amylase preparation C was recorded one test tube was labeled "blank" and the other five as pH 5, pH 6, pH 7, pH 8, and pH 9. 1 ml of appropriate pH starch solution was added to the corresponding essay tubes and 1 ml of 1% starch solution pH 7 was added to the blank tube of which the starch is the substrate solution of the reaction. A fresh dilution of the unknown -amylase was made by mixing 100l of the concentrated enzyme stock with 9.9 of dH20 shaking to mix. The stock and the diluted solutions were kept on ice. All the tubes were retrieved after 10 minutes of pre- incubation step. 1ml of dH20 was added to the blank tube only. To the other five tubes, a timer was set at 12 min. and the count down began immediately after the moment 1ml of dilute -amylase was added to each of the five tubes shaking to mix after which each tube was returned to its appropriate temperature. In order to test maltose 1 ml of maltose color reagent was added to each tube including the blank. The tubes were vortexed after which they were placed in a boiling water bath for exactly 15 mins. Timing was started immediately the maltose color reagent was added to the first tube. The maltose color reagent denatures all of the enzymes hence stopping any enzymatic reaction. The tubes were placed on ice until they cooled to room temperature after which 9 ml of dH20 was added to each tube shaking to mix. Using the blank to zero the spectrophotometer A540nm was measured of all samples and the results were as in the tables 1, 2, 3 and 4 below. Results Table 1 Temperature 40C 230C 370C 650C 1000C Absorbance units (540nm) 0 .05 .18 .06 .05 -amylase activity as a function of temperature Table 2 Temperature 40C Absorbance units(540nm) 230C Absorbance units(540nm) 370C Absorbance units(540nm) 370C Absorbance units(540nm) 1000C Absorbance units(540nm) -amylase A -amylase A Average 0 -amylase B -amylase B Average 0 .01 .15 .24 .43 -amylase C -amylase C Average 0 0 .0 .135 .18 .34 .04 .01 .05 .045 Class data of -amylase activity at different temperatures Table 3 pH pH5 pH6 pH7 pH8 pH9 Absorbance units (540nm) .01 .03 .06 .04 .02 pH as a function of -amylase activity Table 4 pH pH5 pH6 pH7 pH8 pH9 -amylase A -amylase A Average 0.48 0.52 0.3 .09 0.03 Class data of -amylase activity at different pHs Graph 1 Graph of -amylase activity against temperature Graph 2 Graph of -amylase activity against pH Discussion/Conclusion From table 1 on the -amylase activity as a function of temperature the activity of the enzyme was 0 at zero 40C, it increased to 0.05 at 230C and reached the optimum of 0.18 units at 370C and it decreased to 0.06 units at 650C and further decreased to 0.05 at 1000C. Similarly, from table 2 on Class data of -amylase activity at different temperatures the activity of the was 0 at 40C, increased to 0.135 units at 230C and reached its peak of 0.34 units at 370C and decreased to 0.01 at 650C and further decreased to 0.045 units at 1000C. From the above results it is evident that the activity of -amylase is at its highest at body temperature of 370C, a confirmation that enzymes work optimally at this temperature. This is also shown by graph 1 above. Table 3 indicates that at lower pH values, enzyme reactions are lower rising towards neutral Ph and peaks at 7. This fact is illustrated by graph 2 above. References Groves JT "Artificial enzymes. The importance of being selective, 1997 pp. 329-30. Jencks W.P. "Catalysis in Chemistry and Enzymology." 1987, Dover, New York Read More