CCBC - Enzymes and Enzymatic Reactions: synopsis, lab guide and instructors guide to lab exercise


The Biotechnology Curriculum Collection of the California Community Colleges

Enzymes and Enzymatic Reactions: synopsis, lab guide and instructors guide to lab exercise

Synopsis

This exercise is designed to introduce the science major to several major concepts in enzymology and lab skills commonly encountered in biological laboratories. The students are introduced to enzymes and given their general properties and characteristics. The students are then introduced to the enzyme alkaline phosphatase and the reaction that it carries out. The modification necessary to visualize the alkaline phosphatase reaction is explained as well as the necessity for blanks and controls in enzymatic reactions.

An experiment to quantitate the amount of enzyme necessary to carry out the next set of experiments (with the necessary blanks) is laid out for the students. This is an attempt to enhance the students understanding of a real-life laboratory situation where the concentration of an enzyme from a biological extract must be quantitated before use. The students carry out this experiment, graph the results using Cricketgraph™, and interpret the results.

A second set of experiments to test the effects of different environmental conditions (changing temperature, pH and substrate concentration) is introduced to the students. The students, using the results generated in the first experiment, design their own experiment to test the affects of changing one of the three environmental conditions.

These results are also graphed and general conclusions are drawn from the data generated. Specific questions about the results conclude the experiment.

Technical guide 1. Hazards: The product of this reaction, p-nitrophenol, is a potentially hazardous compound and should not be poured down the drain. Collect the contents of the reaction tubes in a sealed glass container and have professionals dispose of it.

2. Labile reagents: The substrate should last for about a week. The enzyme should be made fresh for each lab period and kept on ice until used for maximum activity.

List of Supplies (for a class of 24, working in pairs)

200 or more cuvettes or very clean test tubes, 13 x 100mm

(Fisher # 14-962-10c borosilicate disposable culture tubes make a good and inexpensive alternative to cuvettes and can be reused until they become hard to clean or scratched) wash tubs with soapy water and test tube brushes
12 test tubes racks

6 or more Spectronic 20 or similar spectrophotometers

12 or more micropipetors, 1ml size with tips (or 1ml and 5ml serological pipets, at least 50 of each) clean but need not be sterile

12 or more computers with Cricketgraph™ installed (or graph paper)

10gm of p-nitrophenyl phosphate, disodium salt, Amersham #19587 (800µg/ml in DI H₂0) (this is the substrate, only about 5gm will probably be used per class; store unused portion at 4

1gm of alkaline phosphatase, Amersham #10945 (200µg/ml in DI H₂0) (this is the enzyme, only about 0.2gm will probably be used per class; store unused portion at –20 in a non-frostfree refrigerator)

incubator with water bath (or water bath) set to 37with racks

2 water baths (or hot plate with beaker) set to 50 and 70 with racks

ice bath with racks

thermometers

parafilm and scissors (or 3 vortex shakers)

grease pencils

Kimwipes™ or similar

hazardous waste container

micropipet tip or pipet disposal

2 liters of deionized water

Buffers:

pH8: 500ml of 0.025M sodium tetraborate (borax) plus 205ml of 0.1M HCl

pH9: 500ml of 0.025M sodium tetraborate (borax) plus 46ml of 0.1M HCl

pH10: 500ml of 0.025M sodium tetraborate (borax) plus 183ml of 0.1M NaOH

pH11: 500ml of 0.05M sodium bicarbonate (NaHCO₃) plus 227ml of 0.1M NaOH

pH12: 500ml of 0.05M sodium monohydrogen phosphate (Na₂HPO₄) plus 269ml of 0.1M NaOH

Supplier of enzyme and substrate:

Amersham Life Sciences
2635 S. Charbrook Dr.
Arlington Heights, IL 60006
Customer Service: 800-323-9750
Technical Assistance: 800-341-7543
Fax: 708-437-1640

Instructor guide

This lab exercise is designed to introduce the science major to enzymes. It is a fairly thorough introduction including the concepts of blanks and controls, graphical treatment and interpretation of experimental data and some data manipulation. Techniques utilized include use of the spectrophotometer, micropipetors, and computers with graphical analysis software. It assumes that the students has been introduced to the use of the spectrophotometer, micropipetors, and computers with graphical analysis software in earlier lab exercises. A lab exercise that introduces the spectrophotometer is included in this package. Both the Helms lab manual and the Bloom, Freyer and Micklos lab manual have labs that introduce the use of the micropipets. The Cricketgraph™ graphing software package includes a tutorial.

Modifications to this lab

This lab can be modified fairly easily to include controls. They were omitted for the sake of simplicity in this version to the lab.

In a second lab period, a lab exercise exploring the activity of enzyme inhibitors could logically follow this lab exercise and would be fairly easy to work up using the alkaline phosphatase system.

Tips to make the exercise run more smoothly

1. The reagent addition problem mentioned on page 6, b. 3)

The adding of the substrate to the reaction mixture starts the reaction. Since the students are dealing with 5 different reactions and their 5 blanks and automated spectrophotometers are not likely available in a teaching lab situation, it is impossible to run all of the reactions at once. What the student will have to do is the run each enzyme concentration separately (with its blank) and then move on to the next concentration.

2. Time zero

Take the first spectrophotometer reading immediately after adding the substrate to an experimental tube and make that reading your time zero reading. It is necessary to do this quickly as in the higher enzyme concentration tubes, much of the substrate will be converted to product in 30s or less. Doing the first reading quickly allows the student to see that the first reading is near 0 absorbance.

3. In the Exercise B substrate concentration experiment, use pH 10 buffer. Have the buffer in a bottle labeled so that the students are not able to answer the optimal pH experiment by reading the label of this bottle.

4. Stress student consistency in methodology in setting up the experiments, timing and in taking the Spec. readings. Their results will reflect diligence.

5. Answers to questions on page 9 & 10

a. Blanks are used in ex. A to remove the background reagents from the spectrophotometer readings so that only the change in amount of product is being measured. Also the blanks are controlling for any oxidation of the substrate.

Controls are not directly used in this experiment but are often used to verify that the reagents are working correctly and are invaluable in experiments that are testing an inhibitor of a reaction

b. Because each experimental tube has a different make up and therefore one blank for all of the experimental tubes won’t work.

c. Positive control: Any of the experimental tubes would suffice as a positive control since they all contain all the reagents necessary for the reaction to proceed.

Negative control: The two most important negative controls are:

1) All the reagents necessary except enzyme, i.e. the same composition as the blanks in this experiment. This will show any production of products due to non-enzymatic interactions. However repeating this negative control is not necessary because of the way this experiment is set up.

2) All the reagents necessary except substrate. This tests to make sure that the enzyme is not working on something other than the substrate to make product. This is not likely, so the omission of this control in this experiment is probably justified.

d. Yes, since all experimental tubes contain enzyme (and the same amount of substrate) eventually all of the substrate will be converted into product.

e. Use C₁V₁ = C₂V₂

f. A high concentration of enzyme converts all of the substrate into product early in the experiment. Maximum absorption will be reached at that point.

g. Exercise A determines the volume of enzyme that shows a 2-3 minute linear aborption in this assay. This enzyme volume is necessary to allow the experimenter to visualize the effect of environmental conditions on the reaction. At a high enzyme concentration, the substrate would be changed too quickly into product and the sensitivity of assay would decrease so that the effects of environment on enzyme could be hard to see.

6. Answers to questions on page 14,15 & 16

a. Answer depends on the experiment

1) Temp experiment: same as the 5 experimental tubes except enzyme. The blanks should be treated the same as the experimental tubes. This controls for substrate breakdown/oxidation under different temp conditions.

2) pH experiment: same as the 5 experimental tubes except enzyme. This blanks for the 5 different compositions and controls for substrate breakdown/oxidation under different pH conditions.

3) Substrate concentration experiment: same as the 5 experimental tubes except enzyme. This blanks for the 5 different substrate concentrations and controls for substrate breakdown/oxidation.

b. graph

c. Answer depends on the experiment

1) Temp experiment: Room temp, 37C and usually 50C show strong activity. All except the 70C tube should reach the same absorbance in time, due to the same amount of substrate in each. 70C should denature the enzyme.

2) pH experiment: pH 10 should show optimal activity. Others will show lesser activity as the conditions deviate from optimal pH until the pH is denaturing.

3) Substrate concentration experiment: The greater the amount of substrate the greater the Absorbance. The final absorbances will not be the same.

d. use m= y₂ - y₁/ t₂ - t₁

e. graph of slope vs. the variable

f. statement of how the variable affected the reaction velocity

If you have any questions or suggestions for improvement, Charlie Hoyt can be reached at (619) 421-6700, x5528 or choyt@swc.cc.ca.us

This lab exercise was developed in part with the support of National Science Foundation (Division of Undergraduate Education) grant # DUE 9552290 and California Community College Chancellor’s Office (Curriculum and Instructional Resources Division, Special Projects) grant # FII 95-621-001.

Back to Top of Page

CCBC is operated by Ventura College.

For more information, please contact: jharber@vcccd.net
Tel: (805) 648-8901 Fax: (805) 648-8988
or see: Ventura College Home Page

CCBC HOME PAGE

Back to Previous Page