The Effects of pH, Temperature, Substrate Concentration, and Enzyme Concentration on Reaction Rates

        My hypothesis is that the the pH 6, 55°C, 20 mM, and 4 x 10-7 M buffer has the fastest reaction rate. The controls used in this experiment are the blanks, which contained H2O2 and gualacol.

Results

        In the effect of pH on reaction rate experiment, the buffer with a pH 4 had the highest absorbency (Figure 1), followed by pH 5, pH 6, pH 3, and pH 8. In the effect of temperature on reaction rate experiment, the buffer at 25°C had the highest absorbency (Figure 2), followed by 35°C, 45°C, and 55°C. In the effect of substrate concentration on reaction rate experiment, the buffer with a concentration of 20 mM had the highest absorbency (Figure 3), followed by 10 mM, 7 mM, 5 mM, 2.5 mM, and 1.25 mM. In the effect of enzyme concentration on reaction rate experiment, the buffer with the concentration of 4 x10-7 M had the highest absorbency (Figure 4), followed by 2 x10-7 M, 1 x10-7 M, 5 x10-8 M, and 2.5 x10-8 M.

Table 1.  Effect of pH on reaction rate (500nm)

Figure 1.  Effect of pH on reaction rate (500nm)

Table 2.  The Effects of Temperature on Reaction Rate (500 nm)

Figure 2.  Effect of Temperature on Reaction Rate (500nm)

Table 3.  The Effect of Substrate Concentration on Reaction Rate (500 nm)

Figure 3. The Effect of Substrate Concentration on Reaction Rate (500 nm)

Table 4.  Effect of Enzyme Concentration on Reaction Rate (500 nm)

Figure 4. The Effect of Enzyme Concentration on Reaction Rate (500nm)

Table 5.  Optimum Temperature for Enzymatic Reaction Rates

Figure 5.  Optimum Temperature for Enzymatic Reaction Rates

Discussion

        The reaction rate was found by taking the slope of the linear section of the absorbency versus time graph. In the temperature experiment, no direct relationship can be found between absorbency and reaction rate. The buffer at 45°C had the fastest reaction rate at 0.00195nm/sec even though its absorbency was lower than 25°C (0.0014 nm/sec) and 35°C (0.00166 nm/sec). The buffer at 55°C had the lowest absorbency and slowest reaction rate at 0.00107 nm/sec. In the pH, substrate concentration, and enzyme concentration experiments, a direct relationship can be made between the absorbency and reaction rate. The buffer with the higher absorbency also has the faster reaction rate.

        The optimal temperature for enzymatic reactions is at 45°C. The least optimal temperature is at 55°C. The buffer at pH 4 had the fastest reaction rate making it the optimal pH for peroxidase. The least optimal pH would be at 8 since the graph was very flat and no reaction rate could be measured. The trend in the substrate and enzyme concentration data shows that the least concentrated buffers had the slowest reaction rates. The most concentrated buffers, 20 mM and 4 x 10-7 M, had the highest reaction rate.

        Since the reaction between the enzyme and buffer can occur very quickly, the delay between shaking the tube with parafilm, wiping the outside with a Kimwipe, and putting into the spectrometer could affect the results. The very first readings are important when calculating the slope. To improve the accuracy of the results, the experiment could be carried out for 2 or 3 minutes to observe the full reaction. In most of the graphs, the curve did not level out. This means the reaction was still occurring when we stop collecting data. Therefore, the slope was measure using all 12 points and the reaction rate might have been slower of faster depending on when the curve flat-lined.