edvo-kit # enzyme · pdf filebased on ap biology lab #2 ... pre-lab preparations 16...

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246EDVO-Kit #

Enzyme MicroarraysNew Microtiter Plate-Based on AP Biology Lab #2

Storage:See page 3 for storage instructions.

EXPERIMENT OBJECTIVES:

The objective of the experiment is to demonstratemicroarray technology using enzyme catalysis.

Students will perform an enzyme assay in amicrotiter plate and determine the rate

of a biochemical reaction.

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EDVO-Kit # 246 Enzyme Microarrays

EVT 006276AM

Table of Contents

Page

Experiment Components 3

Experiment Requirements 3

Background Information 4

Experiment Procedures

Experiment Overview 7

Experimental Procedures 8

Enzyme Data Analysis 13

Study Questions 14

Instructor's Guidelines

Notes to the Instructor 15

Pre-Lab Preparations 16

Experiment Results and Analysis 18

Study Questions and Answers 19

Material Safety Data Sheets 21

3

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Component Storage

A Catalase solution FreezerB Hydrogen peroxide, stabilized RefrigeratorC Phosphate buffer, pH 7.2 (conc.) RefrigeratorD Assay reagent, potassium iodide (conc.) RefrigeratorE Acidification solution (conc.) RefrigeratorF Color enhancer (conc.) RefrigeratorG Color developer (conc.) Refrigerator

• Microtiter Plate

Experiment Components

• Automatic Micropipet and tips• Spectrophotometer/microplate reader (optional)• Timers or clock with second hand• Test tubes (13 x 100 mm) for large assay• Distilled water• Ice

RequirementsAll components areintended foreducational researchonly. They are not tobe used fordiagnostic or drugpurposes, noradministered to orconsumed byhumans or animals.

EDVOTEK and TheBiotechnologyEducation Companyare registeredtrademarks ofEDVOTEK, Inc.

This experiment isdesigned for10 groups.

Store ComponentA in the freezer. Allother components

can be stored in therefrigerator.


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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2003, 2004, 2005, 2006 EDVOTEK, Inc., all rightsreserved. EVT 006276AM

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Principles of Enzyme Catalysis

Enzymes as Biological Catalysts

A biological catalyst is used in trace amounts and accelerates the rateof a biochemical reaction without being consumed or transformedduring the reaction. The equilibrium constant of reactions are notaltered by catalysts. Only the rate of approach to equilibrium ischanged.

Reactions in cells are catalyzed by biological catalysts known asenzymes which can accelerate reactions by as much as 1014 to 1020

times. Enzymes function best under mild physiological conditions ofneutral pH, and temperatures of 37°C. Enzymes are generally veryspecific for the reactions they catalyze. Certain enzymes are regulatedby intracellular concentrations of key metabolites that are not directlyinvolved with the reaction they catalyze. Enzymes that are regulatedin this way are termed allosteric.

Measuring Enzyme Activity

The reactant molecule in an enzyme catalyzed reaction is called thesubstrate. The substrate (S) is transformed to product (P). Before theenzyme can transform the substrate it must first bind to it. Only arelatively small portion of the enzyme molecule is involved with sub-strate binding and catalysis. This region is called the active site. Theactive site contains the critical amino acid residues and, if applicable,the prosthetic groups required for activity.

Initial binding is non-covalent and can be in rapid equilibrium. Afterproductive binding has been achieved, the enzyme-substrate complexbegins to generate product which is subsequently released. The freeenzyme (E) can react with additional substrate and this reaction isrepeated rapidly and effectively. The reaction is summarized using asingle substrate, single product in a non-reversible reaction:

E + S → ES → EP → E + P

Catalase is the enzyme used in this experiment. Hydrogen peroxide is atoxic by-product of aerobic oxidation in intermediary metabolism. Allaerobic life forms are capable of enzymatic peroxide detoxification.Catalase (H2O2:H2O2 oxidoreductase) catalyzes the rapid decomposi-tion of hydrogen peroxide by the following reaction:

2 H2O2 → 2H2O + O2 (gas)

Catalase uses the hydrogen peroxide as a hydrogen acceptor anddonor and the catalytic reaction is referred to as catalatic. Theenzyme can also utilize short chain organic molecules such as ethanol,phenols and formate to reduce hydrogen peroxide. This activity is



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called peroxidative. The catalatic activity is preferred in-vitro. Almost allthe cell types in mammals contain catalase, with liver, kidney and erythro-cytes being particularly rich sources.

Catalase has one of the highest catalytic turn over rates known. Over3.6 x 107 molecules of hydrogen peroxide are converted to product by anenzyme molecule per minute.

The appearance of product (P) or the disappearance of substrate (S) canbe measured as a function of time during a reaction. One can measurethe amount of product formed or the decrease in substrate at regularintervals (in this experiment at 30-sec intervals). This quantity can beplotted as a graph. Typical results are shown in Figure 1 which demon-strate the rate of an enzymatic reaction.

An enzymatic reaction measurement is referred to as an assay. At fixedconcentration and reaction conditions, an enzyme reaction rate canincrease by higher substrate concentrations. The probability of forming EScomplexes increases with more substrate molecules present. Generally,the substrate concentration is thousands of times greater than the enzymeconcentration for in vitro kinetic studies. At the early stages of such areaction, the substrate concentration is in great excess and the rate isapproximately linear per unit of time and is termed the initial velocity (v) orinitial rate of the reaction. The characteristics of the enzyme moleculedetermine the initial velocity. It will always remain the same for an enzymeas long as the substrate is present in excess, the products are not inhibitoryand the pH and temperature remain constant.

[S]1 - [S]2

T1 - T2

In the above equation [S]1 is the molar concentration of substrate at someinitial time T1, and [S]2 is the substrate concentration at a later time T2. Notethat the concentration of substrate decreases with time and the concen-tration of product increases with time. Graphically, this can be repre-sented with the substrate concentration plotted on the y-axis and time onthe x-axis. The decrease in the substrate concentration with time willgenerate a curve. The rate of decrease is fastest at the earliest time pointsof the reaction since the substrate concentration is comparatively higher.The rate of decrease diminishes at later times because the substrateconcentration is lower and the reaction is slower (Figure 1). Within shorttime intervals there will be sections of the curve that are approximatelylinear and the rate of the reaction can be measured. At some substrateconcentration, all the enzyme molecules are bound to substrate and areinvolved in some stage of the catalytic reaction. Under these conditionsthe enzyme is saturated with substrate and there is no increase in reactionvelocity.

Time (min.)

Su

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Co

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1 2 3 4 5 6

Figure 1

V =


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EDVO-Kit # 246 Enzyme MicroarraysBa

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Time (min.)30 60 90

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10

20

30

Figure 2

V =

At time 0, there is no product formed. After 30 seconds,10 µmoles havebeen formed; 20 µmoles after 60 seconds; 30 µmoles after 90 seconds.For the initial period, the rate of this reaction could be stated as 20 µmolesof product formed per minute. Typically, less additional µmoles of prod-uct are formed by the second, third and fourth minute (Figure 2). Foreach successive minute after the initial 1.5 minutes, the amount ofproduct formed is less than in the preceding minute.

As the illustration of Figure 2:

30µmoles - 10µmoles = 20 = 0.33 µmoles/sec

90 seconds - 30 seconds 60

In this experiment, a colorimetric assay will be used. It will measure theamount of hydrogen peroxide remaining after catalysis of H2O2 bycatalase in a coupled secondary reaction with iodide (KI).As shown by the equation that follows:

2 I— + 2 H+ + H2O2 → 2 H2O + I2

In this two-step coupled reaction, hydrogen peroxide (substrate) isconsumed and I2 (product) from the secondary non-enzymatic reaction isproduced in equimolar amounts. Catalase (enzyme) catalyzes theconversion of hydrogen peroxide to water and oxygen. To visualize theenzymatic time course reaction, equal volumes of the catalase incuba-tion reaction mixture are transferred at various time points to an acidicsolution of iodide (KI). In this acidic environment, catalase is denaturedand the enzymatic reaction is terminated. In the second reaction whichis the chemical non-enzymatic reaction, the remaining hydrogen perox-ide converts Iodide (I—) to iodine resulting in the generation of a brown-red color that is characteristic of iodine. During the enzymatic reaction,the remaining hydrogen peroxide (substrate) will decrease in the incuba-tion reaction. As a consequence, the amount of brown color due to thegeneration of iodine (I2) in the chemical reaction will also proportionallydecrease.


The initial reaction rate can also be expressed in terms of the appearanceof product. To determine the rate of the reaction, pick any two points onthe straight-line portion of the graph curve (Figure 2). The amount ofproduct formed between two points divided by the difference in timebetween the two points will be the rate of the reaction. It can be ex-pressed as µmoles product/sec.

[P]2 - [P]1

T2 - T1

SAFETY:

Potassiumpermanganate

(KMnO4) is a strongoxidizing agent. It isoften not allowed inschools and is not

used in thisexperiment.



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Experiment Overview

EXPERIMENT OBJECTIVE

The objective is to understand enzyme catalysis.Students will perform an enzyme assay and deter-mine the rate of a biochemical reaction.

WORKING HYPOTHESIS

If the disappearance of substrate can be measuredas a function of time during a reaction, then acorresponding qualitative observation and/orquantitative determination of enzyme rate can bemade.

EACH LAB GROUP REQUIRES:

0.6 ml Reaction cocktail (Solution #2B)0.8 ml Assay solution (Solution #3)

(Do not to contaminate with vessels orpipets that contained peroxide whiledispensing)

50 µl Diluted catalase on ice (Solution #4)200 µl Diluted phosphate buffer (Solution #1)

1 Permanent marker to label tubes2 1 ml pipets (0.1 ml divisions)1 Automatic Micropipet1 Microtiter strip (2 rows)

IN THIS EXPERIMENT:

• Catalase will be added to a buffered solution of hydrogen peroxide.A time course of the reaction will be obtained by removing aliquotsfrom the reaction well every 30 seconds.

• Aliquots removed during the time course reaction will be added to“assay solution for the remaining hydrogen peroxide” wells thatcontain a catalase denaturing solution. To determine the amount ofsubstrate remaining in the time course reaction the remaining hydro-gen peroxide will be measured.

• Hydrogen peroxide that is not catalyzed by the enzyme catalase willoxidize iodide (I—) in a coupled reaction to give the brown-red(Iodine) color. The color intensity increases with the amount ofhydrogen peroxide remaining from the time course reaction. There-fore, in the enzymatic reaction as the incubation time (betweensubstrate and enzyme) increases, there will be a correspondingdecrease in the amount of hydrogen peroxide and therefore adecrease in the intensity of the red-brown color .

• The color intensity may be qualitatively estimated or quantitativelyusing a spectrophotometer.

LABORATORY SAFETY

Gloves and safety goggles should be worn routinelyas good laboratory practice.


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Overview of the Reaction Steps

In this experiment, microtiter plates will be used to preform the assay. Thefirst well, in lane 2, labelled Control, "C" contains 100 µl of bufferedsubstrate (hydrogen peroxide) and 10 µl of dilute phosphate buffer. Noenzyme is added. Ten µl from the "control" well (lane 2) will be added tothe well labelled "0" in lane 1. This establishes the zero time point (highestcolor intensity of iodine conversion due to hydrogen peroxide buffer. Thefirst well labelled "blank" in lane 1 is used to blank the spectrophotometerfor background.

If 30 sec. intervals are too short for students, you can increase timeincrements to 45 sec. or 1 min. This will mean less H202 for the colorreaction and a lighter color.

Wear Safety Goggleand Gloves.



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Microtiter Plate Enzyme Assay

1. Place the microtiter plate on a white piece of paper and with awater resistant pen, line up and label 8 wells in the top row as follows:

1. B (blank)2. 0 min.3. 0.5 min4. 1.0 min5. 1.5 min6. 2.0 min7. 2.5 min8. 3.0 min

A fixed or variable (5 - 50µl) automatic micropipet is required for mostof the steps.

2. In the second row, label the first well for the control “C” and thesecond well for the enzyme reaction “Rxn".

3. Using a fresh micropipet tip, dispense 10 µl of dilute phosphate buffer(Solution #1) to the well labelled Control (“C”) and 100 µl to the welllabelled blank (“B”).

B 0 0.5 1.0 1.5 2.0 2.5 3.0

C Rxn

Microtiter Plate - Catalase Reaction Assay

Control establishes Zero Timepoint. Peroxide (Substrate) / Buffer. No Catalase.

Catalase (Enzyme) Reaction Assay Well Peroxide / Buffer / Catalase. Catalase converts Peroxide into Products. Timepoints: 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 min.

Blank Assay Buffer Phosphate Buffer (used to blank spectrophotometer for background)

Each well (0.5 to 3.0 min.) contains ( I– ) solution and HCl. Enzyme reactions are stopped upon the addition of the timed enzyme reaction in the wells that contain I– and HCl (0.5 to 3.0 min.). Chemical color reaction is initiated to determine amount of substrate (H2O2) remaining.


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4. Using a fresh micropipet tip, dispense 100 µl of Reaction Cocktail(Solution #2B–hydrogen peroxide in phosphate buffer) in the wellslabelled “C” for the control and “Rxn” for reaction well. Discard thetip.

5. Using a micropipet and fresh tip, transfer 100 µl of the Assay solution(Solution #3) to each of the wells in the top row except the blank. Thewells are labelled in the top row (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0).When you have completed these additions, discard the tip.

6. Using a fresh micropipet tip, add 10 µl of the solution from the controlwell (lane 2) to the 0 well in (lane 1). This will be the zero time pointreaction. The zero time point reaction will have the highest amount ofhydrogen peroxide since no catalase was added to the 0 reactionwell. This will also result in the highest amount of color due to theamount of iodide converted to iodine in the color reaction.

7. With a fresh micropipet tip, add 10 µl of diluted enzyme (catalase–Solution #4) to the well labelled “Rxn”. At this point, the enzymaticreaction has been initiated. Immediately start your timer. Discard thepipet tip.

TIMED ENZYME REACTION

8. After 30 seconds from theaddition of catalase, remove10 µl from the Rxn well and addto the well that is labelled "0.5".You may retain the tip andrinse liberally with distilledwater.

9. Using a fresh pipet tip, removean additional 10 µl from thereaction well after an addi-tional 30 second interval andadd to the well labelled “1.0min”. Use a fresh pipet tip orrinse the tip liberally withdistilled water.

Well Diluted Buffered CatalaseBuffer Substrate H2O2 (Solution #4)

(Solution #1) (Solution #2B)

"B" 110 µl 0 µl 0 µl

"C" 10 µl 100 µl 0 µl

"Rxn" 0 µl 100 µl 10 µl

Table 1:Preparation of Wells"B", "Con", & "Rxn"




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10. Using a fresh pipet tip, remove an additional 10 µl from the reactionwell after an additional 30 second interval and add to the welllabelled “1.5 min”. Use a fresh pipet tip or rise the tip liberally withdistilled water.

11. Using a fresh pipet tip, remove an additional 10 µl from the reactionwell after an additional 30 second interval and add to the welllabelled “2.0 min”. Use a fresh pipet tip or rise the tip liberally withdistilled water.

12. Repeat the transfer of 10 µl aliquots from the reaction well afteradditional 30 second intervals and add to the wells labelled “2.5min”, and “3.0 min” respectively.

13. After the addition of the last reaction aliquot, incubate the reactionsat room temperature for 4 minutes to allow color development.

14. A visual examination will determine the rate of the reaction. Alterna-tively, the reaction can be quantified using a microplate reader or alaboratory spectrophotometer that uses cuvettes which hold volumesin the range of 0.1 and 0.2 ml.

Table 2:Preparation and Detection of Well


TIME(min.)

Blank

0

0.5

1.0

1.5

2.0

2.5

3.0

EnzymeAssay #3Solution

I– + HCl buffer

(See Table 1)

100µl

100µl

100µl

100µl

100µl

100µl

100µl

From"C" well(H2O2)

---

---

---

---

---

---

---

---

From"Rxn" well

(Enzyme andSubstrate)

---

10µl

10µl

10µl

10µl

10µl

10µl

10µl

* Requires the use of a spectrophotometer with small curvettes or a microplate reader.

Qualitative orQuantitativeresults (color)


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QUANTITATION OF THE ENZYME REACTION(OPTIONAL - Experiment needs to be adapted for use withspectrophotometer)

Note: This quantitation procedure requires a spectrophotometer thatutilyzes cuvettes that accomodate 0.2 - 0.4 ml samples.

1. To obtain quantitative spectral results, transfer each of thereactions to a cuvette.

2. Zero the instrument with solution labelled B (Blank). Be sure theinstrument is set at 500 nm wavelength. The instrument shouldread 0 absorbance with the blank solution (no color). Read thesamples and record your data.

3. In the case of a single beam spectrophotometer, after setting theabsorbance to zero replace the blank solution in cuvette witheach of the samples to obtain the reading. In between sampleswash the cuvette with distilled water and adjust the absorbanceto zero using the blank.

4. For a dual beam spectrophotometer, leave the blank cuvette inthe blank position and place the second cuvette in the holder toobtain the readings. Again check the blank between samplereadings.

5. Using a clean pipet tip for each sample, transfer the samplesequentially from each well to the corresponding cuvette andmix.

6. Read the absorbance at A 500 and record the absorbancy valuesfor each solution in wells labelled 0.5 to 3.0.




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Enzyme Data Analysis

The reaction rate can be obtained by graphing the absorbancy dataversus time. However, the rate can also be expressed in terms of sub-strate consumed.

1. To express your data in terms of molar concentration of peroxide:

Absorbance x 2.0 = Molarity of hydrogen peroxide in well.ε

ε, the extinction coefficient for this assay system, is 5 x 103.

The molar concentration of either a decrease in substrate or increasein the product can be obtained by dividing the absorbance at A500 bythe molar extinction coefficient. Multiplication by 2.0 (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 provided.

3. Draw the best straight line through the data points. You may noticesome curvature to the data points. This is normal, especially between0 and the first time point, and between later time points. You aremaking a linear approximation.

4. Determine the rate of change in the molarity of hydrogen peroxidewith 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. Determinethe concentration in this way for the next time point.

V = [peroxide1 - peroxide2]

[ time1 - time2 ]

In this experiment, catalase, the enzyme, catalyzes the conversion ofhydrogen peroxide to water and oxygen. In this coupled reaction, themolar concentrations of peroxide (substrate) consumed and I2, theproduct from the non enzymatic reaction are equimolar. To visualize theenzymatic reaction, equal samples of the catalase incubation reactionare transferred at various time points to an acidic solution of potassiumiodide (KI). In the acidic environment catalase is denatured and theenzymatic reaction stops.

In the second reaction, which is a non-enzymatic chemical reaction, theremaining hydrogen peroxide is used at each of the time points to convertthe Iodide (I-) to iodine (I2). Over the time course of the enzymatic reac-tion, the amount of the substrate (hydrogen peroxide) will decrease andconsequently the brown-red color in the chemical reaction will alsodecrease corresponding to the amount of iodine (I2) generated.


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Study Questions

1. What is the enzyme in this reaction?

2. What is the substrate in this reaction?

3. What are the products in this reaction?

4. What is in the assay solution?

5. What is the function of the Potassium Iodine Solution?

6. Explain inhibiting effect of HCl in relation to enzyme structure andfunction.

7. Explain why the color intensity of the peroxide assays decreased withtime?

8. What makes the rate of a reaction of an enzymatic reaction de-crease?

9. Assuming optimal reaction conditions (pH, temperature, etc.) howcould you increase the rate of the reaction other than increasing thesubstrate concentration?

10. The velocity of a catalase reaction was found to increase with in-creasing hydrogen peroxide concentrations as expected. However, athigh peroxide concentrations, the reaction rate decreased andeventually went to zero. What could explain this observation?


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15EDVO-Kit # 246 Enzyme Microarrays

Notes to the Instructor

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Please have the following information:

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OVERVIEW OF LABORATORY INVESTIGATIONS

The "hands-on" laboratory experience is a very important component ofthe science courses. Laboratory experiment activities allow students toidentify assumptions, use critical and logical thinking, and consider alterna-tive explanations, as well as help apply themes and concepts to biologicalprocesses.

EDVOTEK experiments have been designed to provide students theopportunity to learn very important concepts and techniques used byscientists in laboratories conducting biotechnology research. Some of theexperimental procedures may have been modified or adapted to mini-mize equipment requirements and to emphasize safety in the classroom,but do not compromise the educational experience for the student. Theexperiments have been tested repeatedly to maximize a successfultransition from the laboratory to the classroom setting. Furthermore, theexperiments allow teachers and students the flexibility to further modifyand adapt procedures for laboratory extensions or alternative inquiry-based investigations.

ORGANIZING AND IMPLEMENTING THE EXPERIMENT

Class size, length of laboratory sessions, and availability of equipment arefactors which must be considered in the planning and the implementationof this experiment with your students. These guidelines can be adapted tofit your specific set of circumstances.

If you do not find the answers to yourquestions in this section, a variety of re-sources are continuously being added to theEDVOTEK web site.

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ON THE DAY OF THE LAB, PREPARE THE FOLLOWING:

Solution #1 - Dilute Phosphate Buffer pH 7.2 (1x)

1. Pour 19 ml of distilled water into a clean small beaker.2. Transfer 1 ml of phosphate buffer concentrate (Component C) to the

beaker containing the water. Mix. Label the vessel “Dilute PhosphateBuffer”. Store on ice or in the refrigerator. Each group requires 200 µl.

WITHIN 30 MINUTES OF THE LAB, PREPARE THE FOLLOWING:

Solution #2A - 0.12% Hydrogen Peroxide Substrate

1. Transfer 2.7 ml diluted phosphate buffer (Solution #1) to clean 10 mlbeaker or test tube.

2. Using a 1 ml pipet, add exactly 0.3 ml of 1.2% peroxide (Component B)to the 2.7 ml of buffer.

3. Mix.4. Label the vessel “0.12% Peroxide”.5. Let the diluted peroxide cool on ice or in the refrigerator in the dark.

Solution #2B - Enzyme Reaction Cocktail

1. Transfer 6 ml of diluted phosphate buffer (Solution #1) to a clean 15 mlplastic tube.

2. Using a washed or a fresh pipet tip, add 0.3 ml of 0.12% peroxide(Solution #2A) to the buffer.

3. Mix.4. Label the vessel “Reaction Cocktail”.5. Each group will need 0.6 ml.

Solution #3 - Assay Solution (for Colorimetric Assay)

1. Add 15 ml of distilled water to a clean small beaker.2. Add 2.5 ml of potassium iodide solution (Component D).3. Add 2.5 ml of the 0.1 M HCl solution (Component E ).4. Add 2.5 ml of the enhancer solution (Component F).5. Add 2.5 ml of the developer solution (Component G).6. Mix.7. Label the vessel “Assay Solution”. Store in the dark, on ice or in the

refrigerator. Each group requires 0.8 ml.

Pre-Lab Preparations

If the assaysolution is prepared toofar in advance it willbegin to yellow due tooxidation of the iodide.The yellowing willcreate higher blankvalues for thespectrophotometer.

Wear gloves and safetyglasses.

Note:

All glassware needs tobe clean and dry forpreparation of enzymesolutions.

Useful Hint!


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17EDVO-Kit # 246 Enzyme Microarrays

Pre-Lab Preparations

WITHIN 20 MINUTES OF THE LAB, PREPARE THE FOLLOWING:

Solution #4 - Catalase Enzyme

1. With a pipet or graduate cylinder, transfer 2.6 ml of ice cold,diluted phosphate buffer (Solution #1) to the 15 ml plastic tubeprovided.

2. Label “Enzyme”. Keep the tube on ice.3. Carefully add 40 µl of catalase stock (Component A) to the

buffer. The enzyme solution is viscous. Rinse the pipet in the bufferto remove residual enzyme.

4. Cap the tube and gently mix by inverting so that no enzymesolution remains on the bottom.

5. Use within 20 to 30 minutes.6. Each group requires 50 µl.

Spectrophotometer

A well maintained and calibrated spectrophotometer or microplatereader will give good results for your lab activities.

1. Allow the spectrophotometer to warm up one half hour beforethe lab.

2. Set the wavelength at 500 nm. Absorbance will be measured.Color is stable for approximately 1 hour.

EACH LAB GROUP REQUIRES:

0.6 ml Reaction cocktail (Solution #2B)0.8 ml Assay solution (Solution #3)

(Do not to contaminate with vessels orpipets that contained peroxide whiledispensing)

50 µl Diluted catalase on ice (Solution #4)200 µl Diluted phosphate buffer (Solution #1)

1 Permanent marker to label tubes2 1 ml pipets (0.1 ml divisions)1 Automatic Micropipet1 Microtiter strip (2 rows)


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EXAMPLE OF EXPECTED RESULTS:

Absorbancy reading should decrease with time. The pipeting accuracywill affect observed reaction velocities. Representative experiment resultsare presented below:

MINUTES ABSORBANCE MOLARITY*0 1.087 1.86 x 10-3 - (determined experimentally)

0.5 0.915 1.57 x 10-3

1.0 0.811 1.39 x 10-3

1.5 0.693 1.19 x 10-3

2.0 0.594 1.02 x 10-3

* This represents the calculated molarity multiplied by 2.Rate between 0.5 and 1 min is 3.6 x 10-4 Molar/min.

INSTRUCTOR NOTES:

Catalase, the enzyme catalyzes the conversion of hydrogen peroxide towater and oxygen. In this coupled reaction, the molar concentrations ofperoxide (substrate) consumed and I2, the product from the non enzy-matic reaction are equimolar. To visualize the enzymatic reaction, equalsamples of the catalase incubation reaction are transferred at various timepoints to an acidic solution of potassium iodide (KI). In the acidic environ-ment catalase is denatured and the enzymatic reaction stops.

In the second reaction, which is a non-enzymatic chemical reaction, theremaining hydrogen peroxide is used at each of the time points to convertthe Iodide (I-) to iodine (I2). Over the time course of the enzymatic reac-tion, the amount of the substrate (hydrogen peroxide) will decrease andconsequently the brown-red color in the chemical reaction will alsodecrease corresponding to the amount of iodine (I2) generated.

Experiment Results and Analysis

Please refer to the kit insert for the Answers to

Study Questions

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Specific Gravity (H 0 = 1) 2

1 N HCI

1MHCI CAS# 7647-01-0

384° F

No data

1.3

1.2

No data

No data

Water soluble

Colorless

No data

N.D. = No data

N.D. N.D.

Water spray, carbon dioxide, dry chemical powder, or appropriate foam

Wear SCBA and protective clothing to prevent contact with skin and eyes

Emits toxic fumes under fire conditions

09/11/02

Stability

Section V - Reactivity DataUnstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

X None

Acetic Anhydride-alc. Hydrogen Cyanide, AL, Bases, Bronze, CA HCI

Yes Yes Yes

Fumes cause irritation of throat, coughing/choking. Skin contact is corrosive.

Skin irritation, inflammation, ulceration

No data available

Wash with large amounts of water

Wear protective equipment

Observe all Government regulations

Store away from incompatibles

Avoid contact

NIOSH/MSHA approved respirator

Yes No

None

Chemical resistant Safety goggles

Lab coat

Avoid contact - wash thoroughly after handling








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Color Developer

09-16-2002

CAS # 10102-40-6No hazardous components No data

No Data

No Data

No Data

No Data

No Data

No Data

soluble

colorless solution, no odor

Use extinquishing media for surrounding fire


none

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection



No NoneYes None

Rubber Safety goggles

X

Strong oxidizing agents

X

May cause irritation

No data

Eye/skin contact: flush with water Inhalation: remove to fresh airIngestion: wash out mouth with water.

Mop up with absorbent material

Carefully acidify suspension of material to pH with Sulfuric acid. Add a 50%excess of aqueous sodium bisulfite (heat liberated). Observe federal, state and local regulations for disposal

Rubber boots

Avoid contact

none

Yes Yes Yes

Irritation of eye, skin, mucous membranes and upper respiratory tract

No data

Avoid incompatibles

Avoid contact








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Hydrogen Peroxide, Stabilized

Hydrogen peroxide, H2O2 No data 1.2%CAS # 7722-84-1

No data

22.3

1

1.110

No data

No data

Soluble

Colorless liquid, no odor

No data

N.D. = No data

N.D. N.D.

Waterspray

Wear SCBA and protective clothing to prevent contact with skin and eyes.

09-17-2002

at 30°C

Strong oxidizer, contact with other material may cause fire. Container explosion may occur under fire conditions.

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


Excessive heat

Strong acide, aluminum, steel

Toxic oxides of phosphorous

X None

Yes Yes Yes

Acute: Irritates mucous membranes upperrespiratory tract, eyes, skinChronic: May have mutagenic affect

No data

Inhalation: burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea. Irritation.

No data

Ingestion: Wash mouth out with water. Contact physician

Eyes: Flush with water Inhalation: Move to fresh air

Cautiously acidify to pH2 with Sulfuric acid. Add a 50% excess of aqueous

Observe federal, state, and local laws

Store away from incompatibilities

No No

No Chemical fume hood

Rubber Safety goggles

Rubber boots

X

Skin: Flush with water

Mop up with absorbent material and dispose of properly

sodium bisulfate with stirring (heat generated). If no heat is evident, cautiously add until heat is liberated.

Avoid inhalation. Keep away from incompatibilities and combustible material.

NIOSH/MSHA approved








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Assay Reagent, Potassium Iodide

09-19-2002

Potassium Iodide No dataCAS# 7681-11-0

No data

No data

No data

No data

No data

No data

soluble

Clear solution, no odor

No data

Use media appropriate for surrounding fire


Emits toxic fumes under fire conditions.

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection



No None No Chemical safety goggles

Rubber or PVC gloves Safety goggles

X

X

No data

Eye/skin contact: flush with water Inhalation: remove to fresh airIngestion: wash out mouth with water.

Mop up with absorbent material and dispose of properly

Observe federal, state and local regulations

Rubber boots

Avoid contact

Yes Yes Yes

Irritation

No data

Avoid contact and do not store with incompatibles

None

light, air

Strong reducing agents, strong acids, steel, aluminum, alkali metals, brass, magnesium,, Zinc, cadmium, copper, tin, nickel

Hydrogen Iodide

Acute: irritation to mucous membranes, upper respiratory tract, eyes, and skin Chronic: may cause reproductive disorders.

edvo-kit # enzyme · pdf filebased on ap biology lab #2 ... pre-lab preparations 16...

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