The goal of this study was to use spectrophotometry to assess the kinetics of the enzyme glucose oxidase, to analyze the effects of an inhibitor, and also to determine the optimal pH at which glucose oxidase acts. This was done by combining glucose oxidase with a glucose substrate under varying conditions, allowing the reaction to proceed while being read in a spectrophotometer, and using the data read over a time interval to draw conclusions about the varying conditions affecting the rate of reactions to product. Michaelis-Menten and Lineweaver-Burk plots were constructed based on the absorbance values determined from the spectrophotometer readings. The findings of this experiment show that the rate of reaction to products had a maximum velocity of -1.609*10-6M/sec, with a Km value of 0.1796. Other findings show that D-glucal is a competitive inhibitor, and that the optimal pH for glucose oxidase to perform is 6.
The purpose of this research endeavor was to determine the use of enzymes in biological systems, to use a spectrophotometer to assess the kinetics of the enzyme glucose oxidase, as well as to analyze the effects of an inhibitor, and also to determine the optimal pH at which glucose oxidase acts. Enzymes act to increase the speed of a chemical reaction without being changed or used up in the reaction that they catalyze (#1). This is done by lowering the activation energy of the reaction (#1). The Michaelis-Menten equation shows how initial reaction rate and substrate concentration are related. A Lineweaver-Burk plot can also be used to determine kinetics of a reaction, and is generally the reciprical of both sides of the Michaelis-Menten equation. Inhibition can be determined as well from Lineweaver-Burk plots. Competitive inhibition is when an inhibitor of a similar structure to the substrate binds to the active site and vies with the substrate for binding (#2). Non-competitive inhibition is when the inhibitor binds to a site other than the active site and changes the conformation of the active site to inactivate the enzyme (#2). By utilizing the enzyme glucose oxidase to catalyze a reaction, and analyzing that reaction over time through a spectrophotometer, substrate concentration and reaction rate may be determined. By plotting results of spectrophotometric readings, Km and Vmax values may be found. By performing another reaction with D-glucal used as an inhibitor, a supporting graph is used to find out if D-glucal is competitive or non-competitive. Varying the pH of the reaction can also show the optimal pH value that glucose oxidase performs.
Materials and Methods:
pH 7 buffer, glucose oxidase stock, and ABTS/HRP stock is added to wells of a 96-well plate, and water is added as a blank. A concentration gradient of glucose solution is added to the same wells and the samples are read in a spectrophotometer at a wavelength of 725nm every 10 seconds over the course of 3 minutes, and the results are recorded.
pH 7 buffer, glucose oxidase stock, and ABTS/HRP stock is added to wells of a 96-well plate, and water is added as a blank. A concentration gradient of glucose solution is mixed with D-glucal stock solution, and this solution is added to the wells. The samples are read in a spectrophotometer every 10 seconds over the course of 3 minutes and the results are recorded.
pH 4, 5, 6, and 7 buffer are added to wells of a 96-well plate, glucose oxidase stock and ABTS/HRP stock solution is added to these wells, and water is put in the plate as a blank. Glucose stock solution is added to the wells and the plate is immediately read in a spectrophotometer every 10 seconds over three minutes, with the results recorded.
See appendix for all absorbance values and relevant calculations. Using the absorbance values determined from the spectrophotographic readings, and using the extinction coefficient given in the lab of 19000/M/cm, and the pathlength of 1cm, the concentration of the product (D-gluconolactone) can be calculated. Taking the change in product concentration over a time interval yields a reaction velocity, and plotting this vs. the glucose concentration yields the plot below.
Figure 1.1: Michealis-Menten plot of initial reaction velocity vs. substrate concentration, showing the location of Vmax, Vmax/2, and Km values.
By taking the reciprocals of the reaction velocities determined above and plotting them against the reciprocal of the glucose concentration, a Lineweaver-Burk plot may be constructed. From this plot, using the equation of the best fit line, the x and y-intercepts may be calculated, and thus Km can be found by -1/(x-intercept), and Vmax¬ can be found by 1/(y-intercept). This plot is shown on the next page in Figure 2.1.
Figure 2.1: Lineweaver – Burk plot showing the relationship between reaction rate and glucose concentration.
Using the absorbance values determined from the second part of the lab, a Lineweaver-Burk plot may be constructed using the same principles as used on the previous graph. This plot is shown below along with the values from the previous figure, without an inhibitor. The relationship shown can help to determine whether D-glucal is a competitive or non-competitive inhibitor as well as it shows how the rate of reaction is affected by an inhibitor of the glucose oxidase reaction.
Figure 3.1: Lineweaver-Burk plot showing reaction with and without D-glucal, showing that D-glucal is a competitive inhibitor.
By using the absorbance values for varying pH from part three of the lab, as well as the extinction coefficient of 19000 M-1cm-1, the concentrations of the resulting product can be determined. Plotting these concentrations vs. the time taken for their formation yields the plot below. Based on this curve, it is possible to determine at exactly what pH value the enzyme glucose oxidase reacts optimally.
Figure 4.1: Concentration vs. time for varying pH values. The pH 5 line did not yield any change in concentration, possibly due to the accidental omission of glucose oxidase from the well.
Discussion and Conclusions
Plotting the reaction rate vs. glucose concentration as shown in Figure 1.1 gives a curve that follows Michaelis-Menten kinetics because it follows the standard curve for a Michaelis-Menten graph. The Lineweaver-Burk plot (Figure 2.1) was constructed by plotting 1/Vi vs. 1/[glucose]. Using the Lineweaver-Burk plot shows the K¬M value to be .1796, and the Vmax value to be -1.609*10-6. Having a negative value for Vmax is not accurate, and shows there was some experimental error in completing this part of the experiment.
Based on Figure 3.1, the Lineweaver Burk plot showing the reaction with the inhibitor and without the inhibitor, D-glucal is shown to be a competitive inhibitor, because it has a Vmax value that is close to that of the reaction without the inhibitor. The intersection of the two best fit lines occurs close to the y-axis, which is characteristic of competitive inhibition, and not close to the x-axis, which is characteristic of non-competitive inhibition. The results do not show the intersection of the two lines at the y-axis exactly due to the inaccurate absorbance values for the 0.05M glucose concentration data point on the inhibited graph. The absorbance value was lower than anticipated. Possible error in the preparation of the glucose solution, such as insufficient mixing of reactants, could have contributed to this problem. Still, competitive inhibitors do not affect Vmax, and thus D-glucal is a competitive inhibitor.
Based on Figure 4.1 of absorbance vs. time for the differing pH values, the optimal pH for glucose oxidase is pH 6. This is because the best fit line for pH 6 had the greatest value of slope of all the equations for the differing pH values, showing pH 6 to be the most favorable. The greater the slope of the best fit line, the faster the reaction rate. However, we cannot be certain about this value because pH 5 did not yield any absorbance values for our experiment. A possible explanation for the inaccuracy of pH 5 would be the accidental omission of glucose oxidase from the solution.
The enzyme glucose oxidase has many uses outside the laboratory. Commercial uses of glucose oxidase include the following: diabetes monitoring, biofuel cells, food and beverage additive, wine production, oral hygiene, and is also a commercial source of gluconic acid (#3).
Lineweaver-Burk utilizes reciprocals for both the x and y value data points. This distorts the results extrapolated from the plot, because the double reciprocal of the Lineweaver-Burk plot amplifies any experimental error, giving a high error for determining Km and Vmax (#4).
For this experiment, the reactants combine in a similar way to the Cell cycle and ELISA lab. This is because glucose oxidase is similar to the primary antibody used in previous labs. Because glucose oxidase is an enzyme for glucose, it binds to it and in that way it acts like a primary antibody in that it binds to the molecule of interest. Horseradish peroxidase is similar to the secondary antibody from previous labs in that it is bound to the primary antibody (glucose oxidase) and it acts as a bridge to the color changing substrate which in this case is ABTS.
Based on the Lineweaver-Burk plot of the data shown in Figure 8.1 in Appendix 3, the inhibition between the ACE inhibitor peptide and the biologically engineered ACE inhibitor is non-competitive. This data does not support the finding that the biologically engineered inhibitor and the naturally occurring inhibitor are equally capable of inhibiting ACE activity. The reason for this is the difference in the reaction velocities between the engineered and natural inhibitors. The engineered ACE inhibitor is found to be less effective than the natural inhibitor, because the engineered ACE inhibitor has a faster reaction velocity, and is closer to the control, which shows no inhibition.
Table 1.1: Raw data from kinetic reads from the spectrophotometer for all three parts of the laboratory.
Vmax: y-int. = -621502
1/Vmax = -621502
Vmax = 1/-621502
Vmax = -1.609*10-6
KM: x-int. = 1/KM
x-int. = 5.5686152
5.5686152 = 1/KM
KM = 1/5.5686152 = .1796
Figure 8.1: Lineweaver-Burk plot of initial reaction rates of engineered ACE Inhibitor and Natural ACE Inhibitor Peptide, showing the relationship to be non-competitive inhibition.
Sample calculation for changing absorbance into concentration for MM graphs:
Absorbance = ε*c*b
ε = extinction coefficient = 19000 M-1cm-1
b = pathlength = 1cm
c = concentration
Kinetic read 2 (10 sec) of Part 1, 0.1M glucose concentration:
A = 1.476
c = A/ε/b
c = 1.476 / 19000 / 1
c = 7.768*10-5
Sample calculation for Vi for Part 1, 0.1M glucose concentration, over time interval between 10 seconds and 20 seconds, (same calculations also used in Part 2).
Δt = change in time = 10 seconds
Concentration at 10 seconds: 7.768*10-5
Concentration at 20 seconds: 8.479*10-5
Difference = Conc20 - Conc10 = 7.105*10-6
Vi = Difference / Δt = 7.105*10-6 / 10 seconds = 7.105*10-7
1/Vi was used for Lineweaver Burk plots, while Vi itself was used for Michaelis-Menten plots
#1: “Enzyme Kinetics.” Basic Enzyme Reactions. Worthington-Biochem. 27 Oct 2006
#2: “Competitive and Noncompetitive Inhibition.” MIT. 27 Oct 2006
#3: “Glucose Oxidase and Biosensors.” From Biosensors to Food Preservative. InterPro. 27 Oct 2006
#4: “A complete guide to nonlinear regression.” Displaying enzyme kinetic data on a Lineweaver- Burk plot . 1999. GraphPad Software. 27 Oct 2006