Enzymes in cells are very important tothe human body. Enzyme acts as a catalyst that speed up the rate of biochemical reactions that occur in our bodies.The enzyme that will be use in this experiment is Alkaline Phosphatase. The substrate for this enzyme is p-nitrophenyl phosphate, and the product of the overall reaction is p-nitrophenol + Phosphate (PI). The purpose of this experiment is to observe and examine how the reaction rate of product differ when the reaction proceeds with enzyme and without enzyme, with various enzyme concentrations and/or various substrate concentrations. HypothesisThis experiment can be carry out by using the spectrophotometer machine, which will provide the absorbance value needed to create graphs and determine the reaction rate.
Materials and Method:
This section is included in an attached protocol on page
Protocol Lab 6: Enzyme Kinetics
Objective:The purpose of this experiment is to observe and examine how the reaction rate of product differ
when the reaction proceeds with enzyme and without enzyme, with various enzyme concentration
and/or various substrate concentrations.
Sodium carbonate- bicarbonate buffer (0.1M pH 10.0)
P- Nitrophenol Standard (0.05mM)
P- Nitrophenylphosphate substrate solution (5mM)
Serum Alkaline Phosphatase
Numbered test tubes
Part B: Standard Curve of Reaction Product: P-nitrophenol
Label 6 tubes: 1-5 and blank
Using the 0.05mM p-nitrophenol standard, prepare 5 dilutions in buffer with the following concentrations: 0.0025mM, 0.005 mM, 0.01mM, 0.025mM, 0.05mM. Final volume should the 4mL of each dilution.
Blank the instrument at 405nM using 4 mL of buffer
Determine the OD of the 5 dilutions on the spectrophotometer at 405nm and record.
Part C: Rate of Reaction of Hydrolysis of P-Nitrophenyl Phosphate
by Serum Phosphatase as compared to water
1). Prepare the solutions in spectrophotometer cuvettes. Use one cuvette for the samples and another
for the blank. Final volume should be 4mL.
2). Sample 1a; Without enzyme- aID 1.0 mL buffer & 2.6 mL substrate. Mix thoroughly.
3). Sample 1b; Blank- aID 3.6 mL buffer & 0.4 mL of H2O.
4). Use blank sample 1b to zero the instrument att 405nm. Then aID 0.4 mL H2O to the sample 1a, mix
and read at 405 nm to get the time 0 reading. All final volume is 4 mL.
5). Sample 2a; with enzyme- aID 1.0mL buffer & 2.6 mL substrate.
6). Sample 2b; Blank; aID 3.6mL buffer & 0.4 mL serum.
7). Blank the instrument at 405nm. AID 0.4mL serum to sample 2a cuvette, mix and read at 405nm to
get the time 0 reading. Final volume is 4mL.
8). Read sample 2a at 1 minute intervals for 10 minutes and record. Re-blank the instrument after the
Part D: Effect of Serum (Enzyme) Concentration (volume) on the Rate of Reaction of Hydrolysis of
P- Nitrophenyl Phosphate by Serum Alkaline Phosphatase
Label 12 test tubes 1a-6a(samples) and 1b-6b(blanks).
Samples (1a -6a) 2.6mL substrate in each tube and 1.3, 1.2, 1.0, 0.8, 0.6, &0.4 mL buffer in each tube respectively.
Blanks (1b-6b) 3.9, 3.8, 3.6, 3.4, 3.2, & 3.0 mL buffer in each tube and respectively 0.1, 0.2, 0.4, 0.8 & 1.0mL serum in each tube.
Run each tube separately- place blank 1b containing 3.9mL buffer and 0.1mL of serum into a cuvette and blank the instrument at 405nm.
Take sample 1a, aID 0.1 mL serum, mix and read the OD at 405nM to get 0 time reading. Final volume of both sample and blank is 4mL.
Read sample 1a at 1 minute intervals for 10 minutes and record. Re-blank the instrument at the end of the course of the readings.
Run the other pair of tubes (blank and experimental) in the same manner. Do not aID enzyme to a sample cuvette until ready to start the reaction.
Part E: Effect of Substrate Concentration (volume) on the Rate of Reaction of the Hydrolysis of P-
Nitrophenyl Phosphate by Serum Alkaline Phosphatase
Label 7 test tubes 1-6 experimental and 1 test tube for blank
Samples (1-6)- 1.0, 2.0, 2.6, 2.8, 3.0, & 3.2mL substrate in tubes 1-6 and respectively 2.6, 1.6, 1.0, 0.8, 0.6, 0.4 mL of buffer in tubes 1-6.
Blank- 3.6 mL buffer, 0.4 mL serum. Total volume is 4mL.
Blank the instrument at 405 nm. Run each tube separately. Place the sample containing 1.0 mL substrate and 2.6 mL buffer in a cuvette. AID 0.4 mL serum to the sample cuvette, mix and record OD at 405nm to get time 0 reading.
Read the sample at 1 minute intervals for 10 minutes and record the results. Re-blank the instrument at the end of the course of these readings.
Run the other 5 sample tubes in the same manner. Do not aID enzyme until ready to start the reaction.
Attachment:- enzyme lab .docx