Enzymes, Drug Dosage, & Chemotherapy Medical Lab 05
Name(s): Period:
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Date:
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'Background Enzymes
Catalysts are substance'sthat can rate of a reaction withoutbeing change the altered in the Process. EnzymeSare macromolecules that are biological catalysts. All enzymes are proteinsbut not all proteins are enzymes. Thed<.tiVdfiOfit enerᎎ)f a reaction is the amount of energy it takes to start a reaction-the amount of energy it takes to break the bonds of the reactant molecules. Enzymes speed up reactions by lowering the activation energy of the reaction-but without changing the free energy change of the reaction.
Activationenergy (EA) without enzyme
Activationenergy (EA)
g
withenzyme Energy of reactants
Change in free energy Energy of products
aSVÄ$rqte The active site is the part of the
substrate.The enzyme and the substrats foiq a complex called an is generally held together by weak inf@actions.The substrate is then converted into products, and the products are released from the enzyme.
enzyme that binds to
tau_enzynid O Substrates enteractive sä&;enzyme changes shape so its active site
@Substrates held in active site by weak
embraces the substrates (induced fit).
interactions,such as hydrogen bonds and ionic bonds.
Substrates
and speed up a reaction by
complex
• acting as a templatefor substrate • stressing the substrate(S and stabilizing the
OActive site is available for
transitionstate, • providing a favorable
two new substrate
molecules.
microenvironment, • participating directly in the cata lytic reaction.
Enzyme
O
Pro.ducts are
Products Copynght
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dramatically
affected by changes in the and pH and
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temperature. Changes in the
precise shape of an enzyme usually mean the
be as effective.
Products.
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shapesthat are by
enzyme will not
Substrates are Converted into
Released.
three
-dimensional
O Active site (and R groups of its amino acids) can lowerEx
Enzyme-substrate
The activity of an enzyme can be affected by D severalfactors.3Shapeu Enzyme have complicated
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Drug Dosage and Administration The amount
drug administere of of type a the as drug given to a patient isjust as important The amount expressed in weighi of usually drug is the-döéland taken at one time is called while Or volume. For example? it is common for aspirin to be found in dosa#s of 300 mg, cough syrup is is administered at that dosage drug A commonly ml. 5 found in dosages of regular time prescriptionlabel a example, For intervals theßosegeregimem is considered might read: "Take one 250 mg capsule tw•cea dale", ap thedésqg@form.. In addition, a ered in differentforms,called medication adminis be can These could include capsules, creams, liquids,ointments,patches, aerosols,chews, or liquic routes that may just as a few examples. Drugs can also be administered through different include inhaled, topicaBoral, rectal, or through injection. consfderdIlonly factors that must b Drug Facts These are not the Active ingredient (in each tablet) or Purpose Chlorpher,iramine maieatø 2 mg taken into account when prescribing Antihistamine Uses temporanty relievos those symptoms due to hay lever or other upper rosptratoryallecgies: taking a medication. The age of a patien sncozng •runny nose itchy.wateryeyes itchyIhroat sex, ethnicity, weight, previous health Warnings Ask a doctor before se If you have problems,and other drugs that are glaucoma a breathing prouom such as emphysema or chronic bronchitis already being lake must all be troublo urinating duo to an enlarged prostata land provide the optimc Ask a doctor or pharmacist before use If you are taking tranquilizersor sedatives considered in ord o v Whenusing this product dosage for the drug be effective, but Dos ae You may 90t drme•sy avoid alcoholtc drinks alcohol. sedatives. and tranquilizers may increase drowsiness willnot cause adverse side effects. Many brtCf1Dh5 be careful when dnvtng a motor vehicle or operating machinery excitability may occur, especially in children of these factors are included on the "Druc a ha It pregnant or breast-teedlng, ask a health prolesstonalbeforeuse. Facts" label that is required on all overKeep out of reach of children. In case of overdose, get medicathelp or contacta Poison ontrol Center nght away the-counter drugs. An example of the Directions Drug Facts for a common allergy adults and children 12 years and over take 2 tablets every 4 to 6 hours: not more than 12 tablets in 24 hours medication Cdn b*een in the diagram children 6 years to under 12 years take 1 tablet every 4 to 6 hours; the left. This informdfOn includes the not more than 6 tablets in 24 hours children under 6 years ask a doctor osage, directionsfor use, and warnings/ protectfrom excessive moisture Other infortnation store at 20-250 C (68-770 F) uses to consider in order to prevent any Inactive ingredients D&C yellow no. 10, lactose, magnesium stearate, microcryslaJline negative side effects. cellulose, prege'atlnized starch
Importance of Dosage to Chemotherapy
(also caged chemo) is a type of cancer
treatment that uses drugs to destroy cancer cells. Chemotherapy works by stopping or slowing the growth of cancer cells, which grow and divide quickly, but it can also harm healthy cells that divide quickly, such as those that line your mouth and intestines or cause your hair to grow. Damage to healthy cells may cause side effects. The most common side effects are hair loss (alopecia), decreased production of red and Whiteblood cells (myelo- and immunosuppression),and inflammation of the digestive tract lining (mucositis).The majority of chemotherapy drugs target cells that divide quickly. They are designed
by destroying
J2-s0cToCanca (Impdikcd,
2
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damaging cells that divide quickly, such as tumor cells. and cancers that grow?uickly are extremelysensitivetoFor thisreas09, aggressive tumors chemotherqFy. Slowergrowing or solid tumors do not respond as well, due to their slower growth rate, a: CWell as the fact that some cells at the center of solid tumorsare no longer dividing, making chemotherapy completely ineffective. For these types of cancers, chemotherapy is often used combination with surgical removal and/or radiation therapy. Administeringthe in correct dosage of chemotherapy can be tricky. Chemotherapy drugs are cytotoxic, and administering an excessive dose may run the riskof killingthe patient. On the other hand, administering a dose thet is too low may be ineffective on the takget#i tumor. The dosage
weight of the patient.
EXPERIMENT
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Threenew chemotherapy been developed by HASPI Pharmaceuticals. Before they can be considered for use, they tvst bf extensively researched to determine the toxicity level, side effects, and the effeehye dosage. This information must be known in advance to make sure the dosage is high enough to be
effective, but low enough that it does not inhibit normal cellular processes, or even worse, kill normal cells. In this lab, you will be responsible for choosing and testing one of these chemotherapy drugs to determine the toxicity and highest dosage possible before it startsto destroy the function of healthy cells. C
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a v a loz Livercells contain an eryyme called@dtaldseythat is responsibleVr *aking down hydrogen peroxide into oxygen gas and water, which are harmlesst04he body. If catalase
is not effective, hydrogen peroxide would build up in the body and eventually become toxic leading to death. Your team will be looking at how different concentrations of the chemotherapy drugs affect the amount of oxygen gas produced as the livercells (catalase)
break down hydrogen peroxide.
2 HO 0Materials Testtube rack 4 Balloons Liver solution (cells)
4 Large test tubes
Stopwatch Spot plate
catalase Scale Weighing boat 4 Beakers or cups Distilledwater Graduated cylinder Stirrer(spoon)
2 1+0 +0 Chemotherapy Drug A Chemotherapy Drug B C otherapy Drug C ydrogen peroxide 4 PlaStic pipettes
3
Procedure/Direcfions
Yourlab team willbe given tasks, or directions, to perform on the left. Record your que: observations, or required response to each task on the right.
PartA: Chemotherapy Dosage 'Taskt
2
Response a. What drug are you testing?
As a medical research team, choose ONE of the dru s A, B, orc to test. The amount of drug in grams that is added to water represents the concentration. When diluting the drug into a liquid, it is often
represented as a percentage. For example, if I gram of drug is added to 9 ml of water, for a total of 10 ml: Divide 1/10,which equals 0.1. Multiply0.1 x 100 to get the percentage, which
b. Determine the concentration (70)of t} followingamounts of drug added to ma; ml of solution.
is 10%.
0.5 g of dicjg =
soZ
5 g of drug =
3
What isthe highest concentration before liverfunction is greatly impacted?
formulate a
4
Discuss with your team and
a. What is our hypothesis?
;s
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The
to answer thisquestion.
For this experiment, if it takes the liver cells more than 60 seconds to completely fill the
a. What 3 concentrations
We testing?
is your team
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balloon with oxygen, then liver cell function is being impacted. You want to get the time as
close to 60 seconds as possible,but not over.
5
Just as a research feam would, it is up to your team to determine what concentrations you are going to test. Discuss and record your
concentrations to the right. DO NOTforget that you also need to have a control.
b. What is your cdhthpl?
Ohtroj W hou{--L clvv Round 1 Concentrations:
6
Calculate the amount of grams of drug you willneed to add towater for a total of 10 ml of solution. Record your amounts to the right.
ml water = 10r
g drug + =
4
drug +
ml water = 10m ml water = 10r
7
Using the spoon and a beaker/cup. add approximately 4-5 spoonfulsof the drug your group chose to the beaker/cup.
8
Label the 3 remainin beakers/cups with the concentrations bein tested.
9
Using the scale anĂŠweighing boat, measure out the drug with the amojnts needed for each concentration. As you weigh each amount, add it to the beaker/cup labeled with that concentration.
Use a graduated cylinder to measure the amount of water needed to make the 10 ml of total solution for each concentratioa. As you measure each amount. 10 pour it into the beaker/cup labeled with that concentration. Use the spoon to thoroughly mix each solution. Dry off the spoon with a paper towel
between stirsfor each beaker/cup. You shouldhave 10 ml of total solution for each concentration.
15 drops of liver solution to 3 wells of the spot plate. Label 11 Add each of the
wells with the concentration to be tested. If you are wonderirig why you have 4 test tubes, REMEMBER: 12 You also need to test your control... Using a pipette,add 15 drops of each drug solution to its 13 res ective well. Allow at least 5 minutes for the drug solution to diffuse and impact the function of the 14 liver cells. While this is happening, measure out and pour 3 ml of hydrogen peroxide into each of the 3 test tubes. Lay the balloons out next to the test tubes. 15
On a separate shee of paper, create a thatinclu es 8 concentrationsyou are testing and provides space to record your results(time).
Use a plastic pipette to QUICKLY add 5 drops of the 16 liver/drug solution from one of the wells to the first test tube containing 3 ml of hydrogen peroxide. QUICKLY plqce over the test tube and start the stopwatch.
The balloon will oxygen gas released as the 17 catalase breaks down hydrogen peroxide. When
enough oxygen has been released to cause the balloon to stand completely upright,stop the stopwatch and record the time. If the time reaches
3 minutes, stop the time and write "DNF" (did not finish)in the resultsön your data table. 18 Repeat steps 16 and 17 for the remaining drug solutions. Record your results. Now that you have the resultsfor the 3 concentrations you chose to test, it is time to 19 re-evaluate your hypothesis. Summarize your results for your firstreund of testing and revise your hypothesis.
a. Revised hypothesis:
Raupgafgctersz Factor #1:
Clean and rinse out your beakers, test tubes, Trial 1: the with and pipettes. Repeat the experiment Trial 2: 20 best concentration but this time experiment Trial 3• with two factors thä affect enzyme activity. Record all of your results in your data table. Factor #2: Trial l: Trial 2: Trial 3:
Analysis Questions 1, 2. 3. 4. 5. 6.
What are enzymes and how do they work? Describe two factors that affect enzyme activity. What is the substrate used in this experiment? List and explain the enzyme used in this experiment. Create a visual that shows the action of this enzyme (refer back to Figure 8.15). Create a data table that summarizes your results.
7. Wereyou able toget a good idea of what concentration
cell function? Explain why or why not? 8. What can you conclude from your results? 9. Discussat least 3 sources of errorthat cowld have skewed your results. 10. Why is testing like this important for chemotherapy drugs?
to not impo
Discussion Question The following question and corresponding data will be collected in a whole-class discussion . 1. Listthe two factors your team tested (örOceduresteÄ20f xd giscusstheir,effeetsø the textbook or Make sure to support all findings with evid class resource.
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'Analysis Questions 1. What are enzymes and how do they work? Enzymes are macromolecules that are biological catalysts. Enzymes speed up reactions by lowering the activation energy of the reaction, but without changing the reaction's free energy.
2. Describe2 factors that affect enzyme activity. The activity of an enzyme can be affected by several factors. Enzymes have complicated 3-dimensional shapes that are dramatically affected by changes in the pH and temperature. Changes in the precise shape of an enzyme usually mean the enzyme will not be as effective. Changes in pH and temperature can denature the shape of the enzyme.
3. What is the substrate used in this experiment?
The substrate that is used in this experiment would be "drugs" that we were testing with the liver solution because they are reacting wi h the liver in order to create a reaction.
perox
4. List and explain the enzyme used in this experiment.
The enzyme that was used in this experiment would be the liver solution that we combined with the "drugs". Liver contains catalase which is an enzyme that is responsible for breaking down hydrogen peroxide into oxygen gas and water, which are harmless to the body.
5. Create a visual that shows the action of this enzyme.
Substrates enteractive site. enzyme changes shape so rtsecuve Site
embraces the substrates (induced fit)
Substrates
Substrates held in active Site by weak interactions,such as hydrogen bonds and ionic bonds.
Active site (and R groups of its amino acids)can lower
Enzyme-substrate complex
and speed up a reaction by • acting asa template for substrate orientation, • stressing the substrates and stabilizing the transition state, • providing a favorable microenvironment. • participating directly in the catalytic reaction.
ctve site
availabiefor two new substrate moiecules.
Enzyme
O Products are
O Substratesare
Released.
Converted into
Products
Products.
6. Create a data table that summarizes your results. Concentration
Time (Seconds)
00/0
DNF (Did not finish)
5%
DNF (Did not finish)
DNF (Did not finish) DNF (Did not finish)
40 0 0
concentration is safe enough to not impair cell 7. Were you able to get a good idea of what function? Explain why or why not? was safe enough to not impair cell Yes, we were able to get a good idea of what concentration determined that the safest concentration function. At the temperature we were testing at we would be 000 because of the speed of the reaction that took place.
8. What can you conclude from your results? My group was able to conclude that the substrates introduced to the enzyme at 52 celsius were not helpful to the breakdown of hydrogen peroxide. We discovered that the reactions for all concentrations did not finish, but the one with 20% did produce the most gas out of all. This would mean that the drugs are either ineffective or would prove more detrimental to the healthof the average person than not having any drugs introduced into their system at all.
9. Discuss at least 3 sources of error which could have skewed your results. source of error that could have skewed our results would have been that we put the balloon on top of the test tube incorrectly. This could have led to gas escaping through the top of the test tube which would have resulted in inaccurate test times. Another source of error could have been inaccurate measurements of hydrogen peroxide. Some were more than others because we did not take the time to make them all accurate and the same. This could lead to skewed error as we would have calculated the wrong amount of substance that we put into the mixture. A final source of error could have been that we had faulty measurement instruments.This would mean that we would have had incorrect measurements when measuring out the amount of enzyme and substrate that we would be using in the lab. This could lead to skewed error as we would have calculated the wrong amount of substance.
10. Why is testing like this important for chemotherapy drugs? Chemotherapy drugs must be extensively researched to determine the toxicity level, side effects, and the effective dosage. This information must be known in advance to make sure the dosageis high enough to be effective, but low enough that it does inhibit normal cellular processes, or even worse, kill normal cells.