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Experiment Objective: The purpose of this experiment is to understand enzyme catalysis. Students will perform an enzyme assay and determine the rate of a biochemical reaction.
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All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
EVT 910148AM
None of the experiment components have been prepared from human sources.
Experiment Components Requirements Background Information Experimental Procedures Analysis Study Questions Instructor's Guide Pre-Lab Preparations Expected Results Answers to Study Questions Material Safety Data Sheets
All components of this experiment are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
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Experiment Components
This experiment is designed for 10 groups.
Storage: Store component A in the freezer. All other components can be stored in the refrigerator.
Contents A. B. C. D. Catalase solution 1.2% hydrogen peroxide, stabilized Phosphate buffer, pH 7.2, 20x concentrate Assay reagent, potassium iodide, concentrate E. Acidification solution, 0.1 M HCl F. Color enhancer, concentrate G. Color developer, concentrate One 15 ml plastic tube
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
Requirements
Visible wavelength spectrophotometer Test tube racks Timers or clock with second hand Lab permanent markers Test tubes (13 x 100 mm, 10 ml) Beakers Distilled water 5 and 10 ml pipets Pipet pumps or bulbs Linear graph paper 20 - 1 ml pipets, reusable glass or plastic with 0.1 ml divisions, or an automatic pipetor (200-1000 l) and tips (more will be required for Instructor Pre-Lab) Ice
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
BACKGROUND INFORMATION
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
BACKGROUND INFORMATION
ture. The patterns are complex, having bends, twists and spirals. Secondary structure is mainly determined by hydrogen bonds between backbone oxygens, nitrogens and hydrogens. Well known examples of secondary structure include -helices and -pleated sheets. Secondary structure is influenced by the type of amino acids present in that part of the polypeptide chain. The complete three dimensional folding pattern of a polypeptide chain, including all the positions of the amino acid functional groups is called the tertiary structure. The tertiary structure creates the crevices and pockets which enable the protein to bind and react with other molecules. It also gives the protein its shape and affects its solubility. Most importantly, the precise tertiary structure is absolutely necessary for the proteins biological activity. Many proteins consist of several polypeptide chains that are specifically associated with each other by non-covalent and covalent bonds. The three dimensional arrangement of polypeptide chains to each other in a protein is called quaternary structure. The individual polypeptide chains that make up the protein are often called subunits. The subunits of a protein can be identical, similar, or completely different from one another. Different subunits can be responsible for different functions within a protein. Certain proteins also contain, as integral parts of their structure, chemical groups that are not part of the amino acid residues but are absolutely required for biological activity. These groups include small organic molecules, such as certain vitamin derivatives, and certain metal ions. Moieties such as these are called prosthetic groups. A well known prosthetic group is heme. Heme consists of an iron atom coordinated to the nitrogens of a set of organic rings called porphyrin. A protein that contains all its natural structural elements and possesses biological activity is called native. When a protein has been unfolded, it no longer possesses biological activity even though the backbone and the amino acid groups remain intact. Unfolding also causes subunit dissociation if there are no intersubunit covalent links between them. Unfolded, inactive proteins are called denatured. The reactant molecule in an enzyme catalyzed reaction is called the substrate. The substrate (S) is transformed to product (P). Before the enzyme can transform the substrate it must first bind to it. Initial binding is non-covalent and can be in rapid equilibrium. After productive binding has been achieved, the enzyme-substrate complex can now generate product which is subsequently released. The free enzyme (E) can now react with more molecules of substrate (the enzyme has turned
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
BACKGROUND INFORMATION
ES
EP
E+P
The disappearance of S or the appearance of P (or both) can be measured as a function of time. This relationship is the rate of the reaction. The method of measurement is called the assay. At a fixed enzyme concentration and fixed reaction conditions, the reaction rate can be increased by increasing substrate concentrations. The probability of forming more ES complex increases when there are more substrate molecules present. Generally, the substrate concentration is thousands of times greater than the enzyme concentration in kinetic studies performed invitro. At the early stages of the reaction, if the substrate concentration is in great excess, the rate is approximately linear with time and is termed the initial velocity (v). [S]1 - [S]2 ______________ T1 - T2 where [S]1 is the molar concentration of substrate at some initial time, T1, and [S]2 is the substrate concentration at a later time, T2. The reaction rate can also be expressed in terms of the appearance of product: [P]2 - [P]1 ________________ T 2 - T1 Note that the concentration of substrate decreases with time and the concentration of product increases with time. Graphically, this can be represented with the substrate concentration on the y-axis and time on the x-axis. The decrease in the substrate concentration with time will generate a curve. The rate of decrease is fastest at the earliest times of the reaction since the substrate concentration is comparatively high. The rate of decrease diminishes at later times because the substrate concentration is lower and the reaction is slower. Within small time intervals there will be sections of the curve that are approximately linear and the rate can be estimated. The rate of an enzyme reaction cannot be increased indefinitely by continuously increasing the substrate concentration. At some substrate concentration, all the enzyme molecules are bound to substrate and are involved in some stage of the catalytic cycle. Under these conditions the enzyme is saturated with substrate and no further increase in reaction velocity is observed.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
BACKGROUND INFORMATION
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
BACKGROUND INFORMATION
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
EXPERIMENTAL PROCEDURES
EXPERIMENT OBJECTIVE:
The purpose of this experiment is to understand enzyme catalysis. Students will perform an enzyme assay and determine the rate of a biochemical reaction.
LABORATORY SAFETY
Gloves and goggles should be worn routinely as good laboratory practice.
Remember!
Catalase will be added to a buffered solution of hydrogen peroxide. A time course of the reaction will be obtained by removing aliquots from the reaction tube every 30 seconds. These aliquots will be added to separate tubes of assay solution. The assay solution denatures the enzyme, catalase, which destroys its activity. The iodide (I-) in the assay solution is oxidized by any remaining peroxide, producing a red-brown iodine ( I2 ) solution. The color intensity can be quantitated in the spectrophotometer and the rate of the reaction determined. The concentrations of peroxide and enzyme in the reaction are approximately 1.8 milliMolar and 5 nanoMolar respectively.
Wear gloves and safety glasses. Label your tubes at the top with a permanent marker.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
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EXPERIMENTAL PROCEDURES
3.
Remember!
5.
6.
Upon addition of enzyme catalase, the reaction will begin. Start timing the reaction immediately and aliquot 0.3 ml of this mixture to tubes labeled 0.5, 1.0, 1.5 and 2.0 at 30 second intervals.
7.
Enzyme reaction cocktail is added. Hydrogen peroxide that is not catalyzed by the enzyme catalase will oxidize iodide to give a brown-red color. The color intensity increases with the peroxide concentration.
Con Peroxide/Buffer No Catalase Rxn Peroxide/Buffer Catalase
Blank Assay Buffer Phosphate Buffer (used to blank B spectrophotometer for background)
Catalase (Enzyme) Converts Peroxide into Products Timepoints 0.5, 1.0, 1.5 and 2.0 2.0
Assay Tubes
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
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EXPERIMENTAL PROCEDURES
9.
10. With the 1 ml pipet, remove 0.3 ml from the Rxn tube and at 1 minute, add it to tube 1. Mix. 11. With 1 ml pipet, remove 0.3 ml from the Rxn tube and at 1 minute 30 seconds, add it to tube 1.5. Mix. 12. With 1 ml pipet, remove 0.3 ml from the Rxn tube and at 2 minutes, add it to tube 2.0. Mix. Set pipet aside. 13. Wait 4 minutes after your last time point to allow full color development.
Data Collection
Spectral readings can now be taken. Depending on the spectrophotometer, you may be able to insert your test tubes directly into the instrument. Otherwise transfer the entire enzyme reactions in tubes provided by your instructor starting with 0 to 2.0.
TIME (min)
Blank 0 0.5 1.0 1.5 2.0
Assay Solution
3 ml 3 ml 3 ml 3 ml 3 ml 3 ml
Diluted Buffer
0.3 ml -----------
Volume Con
--0.3 ml ---------
Volume Rxn
----0.3 ml 0.3 ml 0.3 ml 0.3 ml
A500
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
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EXPERIMENTAL PROCEDURES
Analysis
The reaction rate can be obtained by graphing the absorbancy data versus time. However, the rate can also be expressed in terms of substrate consumed. 1. To express your data in terms of molar concentration of peroxide:
Absorbance x 11 = Molarity of hydrogen peroxide in Rxn tube. e
e is the extinction coefficient for this assay system and has been determined by your instructor and will be provided. Multiplication by 11 (dilution factor) gives the peroxide concentration in the reaction tube. Scientific notation will make the calculations more convenient. 2. Graph the peroxide concentration on the y-axis versus time on the x-axis. Draw the best straight line through the data points. You may notice some curvature to the data points. This is normal, especially between 0 and the first time point, and between later time points. You are making a linear approximation. Determine the rate of change in the molarity of hydrogen peroxide with time. The rate is equivalent to the slope of the line. Pick a time, go vertically up to the line, then horizontally to the y-axis. Determine the concentration in this way for the next time point. rate = [peroxide]1 - [peroxide]2 | time 1 time 2 |
3.
4.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
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Study Questions
1. Why did you observe bubbles in Step 18 of the experiment? Assume you had boiled the enzyme solution before adding it to the peroxide. Would you expect to see bubbles? What gas do the bubbles contain? Why did the color intensity of your peroxide assays decrease with time? What makes the rate of a reaction of an enzymatic reaction decrease? Assuming optimal reaction conditions (pH, temperature, etc.) how could you increase the rate of the reaction other than increasing the substrate concentration? An active preparation of catalase was exposed to the proteolytic enzyme, trypsin. The catalase preparation was found to be inactive when it was reassayed. Why? Concentrated solutions of catalase have a red color. Why? The velocity of a catalase reaction was found to increase with increasing hydrogen peroxide concentrations as expected. However, at high peroxide concentrations, the reaction rate decreased and eventually went to zero. What could explain this observation? Which of the following generalized enzyme- catalyzed reaction schemes best describes the catalase reaction? a. E + S
2.
3.
4.
5.
6. 7.
8.
ES EP
ES1S2
E + P E + P
b. E + S1 + S2 c. E + S
EP
ES EP1P2 E + P1 + P2
ES1S2
d. E + S1 + S2
EP1P2 E + P1 + P2
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
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Notes:
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM