Name: peroxidase lab: ph and temperature effects
Uploaded: 2019-01-29T11:04:45.000Z
Duration: 2 min 20 s
Channel: JoVE

<ul><li><a href="#" data-time="7">Investigating the Effect of pH and Temperature on Peroxidase Activity
</a></li></ul>

enzyme activity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Investigating the Effect of pH and Temperature on Peroxidase Activity
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To make the extraction buffer for the peroxidase enzyme, mix equal volumes of 0.1 M sodium phosphate monobasic and 0.1 molar sodium phosphate dibasic solutions to achieve a final volume of 500 mL.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Test the resulting solution using a pH meter. The mixture should be between a pH of 6.8 and 7.2.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To extract the turnip peroxidase, use a knife to remove the outer layer of a turnip and then cut the turnip into approximately two cm cubes.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place 30 g of turnip cubes into a blender with 300 mL extraction buffer, and blend the turnip cubes at high speed three times for about 15 s each time.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Filter the homogenized mixture through three layers of cheesecloth, then strain the resulting solution through one coffee filter.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Store the filtrate in a 500 mL bottle, and immediately place the bottle in the refrigerator.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, to make the enzyme substrate solutions, first add 15 mL 3% H<sub>2</sub>O<sub>2</sub> to 435 mL distilled water to dilute the H<sub>2</sub>O<sub>2</sub> to a 0.1% concentration.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To prepare the color-changing reactant, dissolve 0.2 mL concentrated guaiacol in 100 mL of isopropyl rubbing alcohol, and store this guaiacol solution in the refrigerator.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, to make solutions for the different pH conditions, add 100 mL distilled water to six 250 mL beakers, and place the buffer capsule contents with pH of 3, 4, 5, 6, 7, and 8 into separate beakers.
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<div class="section-question"><h4 class="question-sect">What are enzymes?</h4><p>Enzymes are biological catalysts that increase the speed and efficiency of biochemical reactions. Most enzymes are proteins but certain ribonucleic acid molecules also have catalytic properties.
</p></div><div class="section-question"><h4 class="question-sect">How do enzymes catalyze reactions?</h4><p>Enzymes catalyze reactions by lowering the activation energy of a reaction.
</p></div><div class="section-question"><h4 class="question-sect">What are catabolic and a...

Enzyme Activity

Investigating the Effect of pH and Temperature on Peroxidase Activity
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peroxidase lab: ph and temperature effects

<p class="jove_content">Peroxidase activity can be tested by changing pH and temperature in a controlled lab setup. In this experiment, students prepare turnip peroxidase and compare how the enzyme reacts under different conditions. The focus is on how pH and temperature can affect enzyme performance.</p>

<p class="jove_content">To make the extraction buffer, equal volumes of 0.1 M sodium phosphate monobasic and 0.1 M sodium phosphate dibasic are mixed to a final volume of 500 mL. The solution is checked with a pH meter. It should fall between pH 6.8 and 7.2. This buffer helps keep the enzyme sample stable during extraction.</p>

<p class="jove_content">Turnip peroxidase is then extracted from fresh turnip tissue. The outer layer of a turnip is removed with a knife, and the turnip is cut into about 2 cm cubes. Next, 30 g of turnip cubes are blended with 300 mL of extraction buffer at high speed three times for about 15 s each time. The homogenized mixture is filtered through three layers of cheesecloth and then through one coffee filter. The filtrate is stored in a 500 mL bottle and placed in the refrigerator right away.</p>

<p class="jove_content">The substrate solutions are prepared separately. First, 15 mL of 3% H2O2 is added to 435 mL of distilled water to make a 0.1% hydrogen peroxide solution. Then 0.2 mL of concentrated guaiacol is dissolved in 100 mL of isopropyl rubbing alcohol. This guaiacol solution is also stored in the refrigerator. To test the effect of pH, 100 mL of distilled water is added to six 250 mL beakers, and buffer capsule contents with pH values of 3, 4, 5, 6, 7, and 8 are placed in separate beakers.</p>

Peroxidase Lab: pH and Temperature Effects

peroxidase-lab-ph-and-temperature-effects

Peroxidase activity can be tested by changing pH and temperature in a controlled lab setup. In this experiment, students prepare turnip peroxidase and compare how the enzyme reacts under different conditions. The focus is on how pH and temperature can affect enzyme performance.<br /><br />To make the extraction buffer, equal volumes of 0.1 M sodium phosphate monobasic and 0.1 M sodium phosphate dibasic are mixed to a final volume of 500 mL. The solution is checked with a pH meter. It should fall between...

Peroxidase Activity: pH and Temperature

Peroxidase activity changes with pH and temperature in this turnip enzyme experiment. The reaction uses guaiacol, a color-changing indicator that becomes more yellow-orange as the enzyme reaction continues, so color intensity shows how fast the reaction is moving.<br /><br />First, a baseline peroxidase reaction is set up. Distilled water, guaiacol, and 0.1% H2O2 are added to make the substrate. A separate tube contains distilled water and turnip peroxidase. After the two tubes are mixed, the color is...

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Prepare turnip peroxidase and test how pH and temperature affect enzyme activity using buffer solutions, hydrogen peroxide, and guaiacol in this high school lab.

Peroxidase Lab: pH and Temperature Effects | JoVE K12

<p>European explorers had adequate knowledge, skills and equipment, but why didn&#39;t they reach the New World until the end of the 15th century? You&#39;ll be surprised to learn that the answer to this question is related to the small biological molecules called enzymes, that act as catalysts. </p>

<p>What is a catalyst? It is a substance that enables a reaction to occur faster, using less energy. If you plotted a graph of the energy needed for an uncatalyzed biological reaction, it would look something like this. This height represents the activation energy, which is the minimum amount of energy needed for the reaction to proceed. Enzymes, working as a catalyst, provide an alternate reaction pathway, with a lower activation energy. </p>

<p>So, how do enzymes actually work? Most enzymes are proteins. The part of their structure which interacts with the molecules they catalyze, is called the active site, which has affinity for only certain substrates and won&#39;t bind with others. When the correct substrates enter the active site, the enzyme changes its shape to attain a higher affinity configuration. This transition state facilitates the reaction and converts the reactants into products. The enzyme, however, has a lower affinity for the products so it releases them and returns to its original shape, ready to repeat the same process. This is an example of a catabolic enzyme because it took a single substrate and broke it to form multiple products. The opposite of this, is the anabolic enzyme, which takes two substrates and joins them together to make a larger product. </p>

<p>There are numerous other ways that enzymes function. For instance, this enzyme transfigures a single substrate into a new shape. Whereas this one, transfers part of one substrate, onto another. Some enzymes cannot catalyze reactions on their own and require cofactors or coenzymes in order to catalyze their reaction. </p>

<p>So, you might be wondering, how does all this relate to the 15th century voyages? At that time, sailors didn&#39;t have access to fresh produce containing vitamin C, a coenzyme essential for collagen synthesis. Because of that, the sailors would develop scurvy, a vitamin C deficiency disease, which was often fatal. They solved this problem by pickling vitamin C-rich fruits and vegetables. Pickling reduced the pH of the food, which prevented the activity of microbial enzymes, responsible for rotting them. Therefore, destruction of microbial enzymes and the saving of an essential coenzyme, played a major role in the success of long expeditions. </p>

<p>In this lab, you can quantify how different conditions affect enzyme function, by assessing the reaction rate, the amount of product made, per unit time. You&#39;ll first measure the baseline reaction rate at a standard room temperature and neutral pH. The rate will increase at the beginning until all of the enzyme molecules are completely saturated with the substrate. And then, the catalysis will reach a steady level as the enzymes are busy continuing the cycle of binding substrate, catalysis and release. Once the baseline reaction rate has been established, it can be used as reference point for other conditions. The enzyme that you&#39;ll work with in this lab is turnip peroxidase. You&#39;ll evaluate its activity under varying values of temperature and pH.</p>


factors that shape enzyme function

<p class="jove_content">Enzymes are biological catalysts that help living things run biochemical reactions faster. They lower activation energy, which is the energy needed to start a reaction. Enzymes are not used up in the process, so they can keep working again and again. Most enzymes are proteins, although some ribonucleic acid molecules also have catalytic activity.</p>

<p class="jove_content">Each enzyme usually binds only one or a few specific substrates. This selectivity depends on the enzyme’s active site, which fits its substrate like a matching shape. When the substrate binds, the enzyme changes shape slightly and makes the reaction easier. After the products form, the enzyme releases them and returns to its original shape.</p>

<p class="jove_content">Enzyme reactions are often grouped as catabolic or anabolic. Catabolic enzymes break larger molecules into smaller products. Lactase is a catabolic enzyme that splits lactose in dairy products into galactose and glucose. Many people lose most of their lactase by early childhood, which can lead to lactose intolerance. Anabolic enzymes build larger molecules from smaller parts. DNA polymerase joins nucleotides to make DNA, and glycogen synthase links glucose molecules to form glycogen. Insulin helps regulate glycogen synthase, so people with little or no insulin may need treatment to make glycogen normally.</p>

<p class="jove_content">Enzyme activity depends on conditions such as pH and temperature. Many active sites contain acidic or basic amino acids, so changes in pH can alter charge and make substrate binding harder. Pickling uses this idea by lowering pH and slowing microbial enzyme activity. Temperature also matters because each enzyme has an optimal range. Low or moderate shifts may slow the enzyme, but high temperatures can denature it and cause lasting damage. Cooking meat and refrigerating food both reduce harmful microbial activity in this way. Extremophiles live in harsh environments and have enzymes that still work there. Thermus aquaticus is one example, and its Taq polymerase stays stable in the high heat used for PCR, a method that copies DNA by cycling between hot and cooler temperatures.</p>

<p class="jove_content">Some enzymes also need coenzymes or cofactors. Coenzymes are biomolecules that help the reaction and are not consumed. They often carry electrons, atoms, or functional groups, and many B vitamins serve this role. Coenzyme A, the active form of pantothenic acid, is needed by many mammalian enzymes. Cofactors are usually inorganic substances, such as ions. DNA polymerase, for example, requires Mg2+ or Mn2+ for DNA replication and for correcting some mistakes.</p>

<p class="jove_content">Other molecules can change enzyme activity without being required for the reaction. Activators increase activity, while inhibitors decrease it. These molecules may bind at the active site or at allosteric sites, which are different locations on the enzyme. Some inhibitors compete with the substrate for the active site, while others act in a non-competitive way by binding elsewhere. Inhibitor binding can also be reversible or irreversible. Reversible inhibitors can detach and let the enzyme work again, but irreversible inhibitors permanently damage the enzyme. Scientists use enzyme inhibitors as antibiotics and as chemotherapeutic agents to limit cancer cell division.</p>

<p class="jove_content">Scientists measure enzyme function by looking at reaction rate, or how fast product is made. A faster rate means the enzyme is working more efficiently. Reaction velocity is measured by tracking product concentration over time, usually from the start of the reaction until the enzyme becomes saturated with substrate and the rate levels off. Researchers use that baseline as a control when comparing different treatments. Reaction rates can be reported as relative or absolute values. Relative rates compare conditions, while absolute rates give a direct quantitative measure, much like comparing race rankings versus miles per hour.</p>

<p class="jove_content">Because enzymes are so useful, researchers also engineer them for special tasks. Protein engineering uses recombinant DNA technology to change amino acid sequences and adjust enzyme shape or active site binding. This approach has helped create sensitive diagnostic tests, as well as tools for bioremediation and waste management.</p>

Factors That Shape Enzyme Function

Biological Catalysts

All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require ...

Test your understanding of Enzyme Activity with this Biology assessment quiz on JoVE Lab Manual. Covers key concepts through video-based questions designed to evaluate student learning.

Enzyme Activity in Biology - Assessment Quiz | JoVE Lab Manual

Measure turnip peroxidase activity using guaiacol, then compare reaction rates across pH 3-8 and temperatures from ice bath to 60 °C.

Peroxidase Lab: pH and Temperature Effects

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What are enzymes?

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