Examining the Effect of Various Enzyme and Substrate Concentration on the Rate of Product Formation 

Introduction:

            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 (P_I). 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.

Materials:

–         
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

–         
Spectrophotometer (405nm)

 

Part B: Standard Curve of Reaction Product: P-nitrophenol

1.)   
Label 6 tubes: 1-5 and blank

2.)   
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.

3.)   
Blank the instrument at 405nM using 4 mL of buffer

4.)   
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- add 1.0 mL buffer & 2.6 mL substrate. Mix thoroughly.

3). Sample 1b; Blank- add 3.6 mL buffer & 0.4 mL of H₂O.

4). Use blank sample 1b to zero the instrument att 405nm. Then add 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- add 1.0mL buffer & 2.6 mL substrate.

6). Sample 2b; Blank; add 3.6mL buffer & 0.4 mL serum.

7). Blank the instrument at 405nm. Add 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

      final reading.

 

Part D: Effect of Serum (Enzyme) Concentration (volume) on the Rate of Reaction of Hydrolysis of

P- Nitrophenyl Phosphate by Serum Alkaline Phosphatase

1.)   
Label 12 test tubes 1a-6a(samples) and 1b-6b(blanks).

2.)   
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.

3.)   
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.

4.)   
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.

5.)   
Take sample 1a, add 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.

6.)   
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.

7.)   
Run the other pair of tubes (blank and experimental) in the same manner. Do not add 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

 

1.)   
Label 7 test tubes 1-6 experimental and 1 test tube for blank

2.)   
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.

3.)   
Blank- 3.6 mL buffer, 0.4 mL serum. Total volume is 4mL.

4.)   
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. Add 0.4 mL serum to the sample cuvette, mix and record OD at 405nm to get time 0 reading.

5.)   
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.

6.)   
Run the other 5 sample tubes in the same manner. Do not add enzyme until ready to start the reaction.

Attachment:- enzyme lab .docx