The reason for this experiment is to determine the activity of the E. coli enzyme Beta-glactosidase. We do this by determining the rate of the enzyme-catalyzed reaction in a preparation with lactose, where the rate of reaction is directly proportional to the amount of enzyme present which is its activity. To be able to do this we used a kinetic assay which allowed us to constantly observe the enzyme reaction by reading the photometer of absorbance increase over the duration of 5 minutes. Enzymes which are typically proteins are biological catalysts. They perform a significant function in biological systems by catalyzing chemical reactions. They do this by binding to the substrate and lowering its activation energy. After the reaction has occurred and the substrate is converted into the product, the enzyme will release and is able to be used again. In this experiment Beta-galactosidase hydrolyzes o-nitrophenyl -B- galactoside (ONPG), a substrate that mimics lactose, converting it into ONP.
Methods:
First, you add 0.5mL of 2.5 mM ONPG and 4.0mL of 0.08M phosphate buffer pH 7.7 to a series of 6 labeled tubes. For Tube 1, add 0.5mL of Enzyme Dilution Buffer (EDB), mix and use as the blank to zero the spectrophotometer. For tube 2, add 0.5mL of undiluted Beta-Glactosidase (B-Gal) solution, quickly cover with Parafilm, mix by inverting, then pour into a cuvette and place it in the colorimeter. You quickly record the first A420nm reading and put it down as time = 0. Then record the A420nm value every 30 seconds for 5 minutes and then one more time after finishing the other tests to get its max change in A420nm, where the reaction is basically over and all substrates are converted into its product. For tube 3, prepare a 2-fold dilution of the enzyme by adding 0.5mL of enzyme stock solution to 0.5mL of EDB. Then adding the enzyme dilution to the tube and take the A420nm readings every 30 seconds for 5 minutes. For tube 4, prepare a 5-fold dilution of the enzyme by adding 0.2mL of enzyme stock solution to 0.8mL of EDB. Then adding the enzyme dilution to the tube and recording the A420nm readings every 30 seconds for 5 minutes. For tube 5, prepare a 10-fold dilution of the enzyme by adding 0.1mL of enzyme stock solution to 0.9mL of EDB. Then adding the enzyme dilution to the tube and recording the A420nm readings every 30 seconds for 5 minutes. For tube 6, prepare a 20-fold dilution of the enzyme by adding 0.05mL of enzyme stock solution to 0.950mL of EDB. Then adding the enzyme dilution to the tube and recording the A420nm readings every 30 seconds for 5 minutes.
Results:
Figure 1: Data Table
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Enzyme Predilution:
0
0
2-fold
5-fold
10-fold
20-fold
Volume of EDB
0
0
0.5
0.8
0.9
0.95
Volume of Undiluted Enzyme
0
0
0.5
0.2
0.1
0.05
Undiluted Enzyme Volume Added to Reaction
-
0.5
-
-
-
-
EDB
0.5
-
-
-
-
-
Diluted Enzyme Volume Added to Reaction (mL)
0
0
0.5
0.5
0.5
0.5
Vp (volume of undiluted enzyme added, mL)
0
0.5
0.25
0.1
0.05
0.025
Volume (mL) Substrate Added (2.5mM ONPG)
0.5
0.5
0.5
0.5
0.5
0.5
Micromoles of ONPG in Each Eeaction
1.25
1.25
1.25
1.25
1.25
1.25
Absorbance at 420nm in minutes:
0
-
0.05
0.039
0.023
0.021
0.03
0.5
-
0.113
0.071
0.036
0.027
0.033
1
-
0.171
0.106
0.05
0.035
0.035
1.5
-
0.233
0.138
0.063
0.042
0.038
2
-
0.289
0.172
0.076
0.048
0.041
2.5
-
0.347
0.204
0.089
0.055
0.044
3
-
0.4
0.238
0.103
0.062
0.047
3.5
-
0.454
0.269
0.116
0.069
0.049
4
-
0.504
0.302
0.129
0.075
0.052
4.5
-
0.55
0.332
0.142
0.082
0.054
5
-
0.593
0.363
0.155
0.09
0.057
Correlation coefficient for X Y plot trendline
-
.9974
.9998
1
.9996
.9985
Slope
.1094
.0651
.0264
.0137
.0054
Maximum change in A420 for Tube 2
1.039
-
-
-
-
Beta-Glactosidase activity in "U"(micromoles of ONP formed per min per mL)
-
0.302
0.304
0.317
0.337
Figure 2: Data Graph
Figure 3: Absorbance Graph Tube #2
Figure 4: Absorbance Graph Tube #3
Figure 5: Absorbance Graph Tube #4
Figure 6: Absorbance Graph Tube #5
Figure 7: Absorbance Graph Tube #6
Figure 8: Raw Data Graph
Figure 9: Calculations
Raw Activity
Tube 2: Tube 3: Tube 4:
(0.1094)(1.25/1.039) = .132 (0.0651)(1.25/1.039) = 0.0783 (0.0264)(1.25/1.039) = 0.0318
Tube 5: Tube 6:
(0.0137)(1.25/1.039) = .0165 (0.0054)(1.25/1.039) = .00650
Enzyme Activity
Tube 2: Tube 3: Tube 4:
[(0.1094)(1.25/1.039)] = 0.263 [(0.0651)(1.25/1.039)] = 0.313 [(0.0264)(1.25/1.039)] = 0.318
0.5 0.25 0.1
Tube 5: Tube 6:
[(0.0137)(1.25/1.039)] = 0.330 [(0.0054)(1.25/1.039)] = 0.260
0.05 0.025
Enzyme Activity Mean = .297
VP (mL)
Tube 3 = 0.5 x 0.5ml = 0.25mL
Tube 4 = 0.5 x 0.2mL = 0.1mL
Tube 5 = 0.5 x 0.1mL = 0.05mL
Tube 6 = 0.5 x 0.05mL = 0.025mL
Conclusions and Interpretation
The average Beta-Glactosidase Enzyme activity is 0.306 µmoles of ONP formed per minute per mL.
