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">探究pH和温度对过氧化物酶活性的影响
</a></li></ul>

<ul class="lab-items"><li data-lab-item="1">
								<div class="ingredient" data-value="试管">试管</div>
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								<div class="ingredient" data-value="冰块（1/2 托盘）">冰块（1/2 托盘）</div>
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								<div class="ingredient" data-value="pH 计/pH 试纸">pH 计/pH 试纸</div>
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								<div class="ingredient" data-value="刀">刀</div>
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								<div class="ingredient" data-value="奶酪布（0.5 米）">奶酪布（0.5 米）</div>
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								<div class="ingredient" data-value="咖啡过滤器">咖啡过滤器</div>
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								<div class="成分" data-value="0.1 M 磷酸钠 二元 soln （250 mL）">0.1 M 磷酸钠 二元 soln （250 mL）</div>
								<div class="成分量">
									<span class="amount" data-value="-1">-1</span>
									<span class="unit" data-value="取决于实验室大小">取决于实验室大小</span>
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								<div class="成分" data-value="0.1 M 磷酸二氢钠 （250 mL）">0.1 M 磷酸二氢钠 （250 mL）</div>
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									<span class="amount" data-value="-1">-1</span>
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								<div class="ingredient" data-value="过氧化氢 （3%） （15 mL）">过氧化氢 （3%） （15 mL）</div>
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									<span class="unit" data-value="取决于实验室大小">取决于实验室大小</span>
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								<div class="ingredient" data-value="愈创木酚 （浓缩液， 1-3mL）">愈创木酚 （浓缩液， 1-3mL）</div>
								<div class="成分量">
									<span class="amount" data-value="-1">-1</span>
									<span class="unit" data-value="取决于实验室大小">取决于实验室大小</span>
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								<div class="ingredient" data-value="异丙基摩擦酒精 （100mL）">异丙醇摩擦酒精 （100mL）</div>
								<div class="成分量">
									<span class="amount" data-value="-1">-1</span>
									<span class="unit" data-value="取决于实验室大小">取决于实验室大小</span>
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								<div class="成分" data-value="pH 缓冲胶囊/pH 值 （pH 范围 - 3-8）">pH 缓冲胶囊/pH 值 （pH 范围 - 3-8）</div>
								<div class="成分量">
									<span class="amount" data-value="-1">-1</span>
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酶活性

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">探究 pH 和温度对过氧化物酶活性的影响
<button class="expand">展开</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">为制备过氧化物酶提取缓冲液，将等体积的 0.1 M 磷酸二氢钠溶液与 0.1 M 磷酸氢二钠溶液混合，最终体积为 500 mL。
</div></li><li data-sub-note-item="1-2"><div class="lab-step">使用 pH 计检测所得溶液，混合液的 pH 应在 6.8 至 7.2 之间。
</div></li><li data-sub-note-item="1-3"><div class="lab-step">为提取芜菁中的过氧化物酶，使用刀具去除芜菁外层，然后将芜菁切成约 2 cm 的立方块。
</div></li><li data-sub-note-item="1-4"><div class="lab-step">将 30 g 芜菁块放入搅拌器中，加入 300 mL 提取缓冲液，以高速分三次搅拌，每次约 15 秒。
</div></li><li data-sub-note-item="1-5"><div class="lab-step">将匀浆混合物通过三层纱布过滤，再将滤液通过一张咖啡滤纸进一步过滤。
</div></li><li data-sub-note-item="1-6"><div class="lab-step">将滤液储存于 500 mL 瓶中，并立即放入冰箱冷藏。
</div></li><li data-sub-note-item="1-7"><div class="lab-step">接下来，为制备酶底物溶液，先将 15 mL 3% H<sub>2</sub>O<sub>2</sub> 加入 435 mL 蒸馏水中，使 H<sub>2</sub>O<sub>2</sub> 稀释至 0.1% 浓度。
</div></li><li data-sub-note-item="1-8"><div class="lab-step">为制备显色反应物，将 0.2 mL 浓愈创木酚溶解于 100 mL 异丙醇中，并将此愈创木酚溶液存放在冰箱中。
</div></li><li data-sub-note-item="1-9"><div class="lab-step">然后，为制备不同 pH 条件下的溶液，向六个 250 mL 烧杯中各加入 100 mL 蒸馏水，并将 pH 分别为 3、4、5、6、7 和 8 的缓冲胶囊内容物分别放入不同的烧杯中。
</div></li></ul></li></ol>

<div class="section-question"><h4 class="question-sect">酶是什么？</h4><p>酶是生物催化剂，能够提高生化反应的速度和效率。大多数酶是蛋白质，但某些核糖核酸分子也具有催化特性。</p></div><div class="section-question"><h4 class="question-sect">酶如何催化反应？</h4><p>酶通过降低反应的活化能来催化反应。</p></div><div class="section-question"><h4 class="question-sect">什么是分解代谢和合成代谢反应？</h4><p>大多数酶促反应可分为分解代谢或合成代谢两类，这两类反应的功能相反。介导分解代谢反应的酶将较大的物质分解为多种产物，而合成代谢酶则将多种物质结合形成单一产物。</p></div><div class="section-question"><h4 class="question-sect">环境条件的变化如何影响酶活性？</h4><p>酶所处环境的条件变化可能导致酶无法正确折叠，或在初始折叠后发生形状改变，亦或改变活性位点的化学性质，这些因素均会阻碍酶与底物的有效结合或催化反应的进行。<...

探究pH和温度对过氧化物酶活性的影响  
为制备过氧化物酶提取缓冲液，将等体积的……混合

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细胞结构与功能

Enzyme 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

enzyme activity

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...

lab manual sub articles

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>欧洲探险家虽然具备充足的知识、技能和装备，但为何直到15世纪末才抵达新大陆？你可能会惊讶地发现，这个问题的答案与一类名为酶的小型生物分子有关，酶能够充当催化剂。</p>

<p>什么是催化剂？催化剂是一种能够使反应更快进行并减少所需能量的物质。如果绘制出非催化生物反应所需的能量图，结果会类似于这样。这个高度代表活化能，即反应进行所需的最低能量。酶作为催化剂，提供了一条活化能更低的替代反应途径。</p>

<p>那么，酶究竟是如何发挥作用的呢？大多数酶是蛋白质。其结构中与催化分子相互作用的部分称为活性位点，该位点只对特定的底物具有亲和力，而不会与其他底物结合。当正确的底物进入活性位点后，酶会改变其构象，以形成亲和力更高的状态。这种过渡态有助于反应的进行，并将反应物转化为产物。然而，酶对产物的亲和力较低，因此会释放产物并恢复至原始构象，准备重复相同的过程。这是一种分解代谢酶的实例，因为它将单一底物分解为多个产物。与此相反的是合成代谢酶，它将两个底物结合在一起，生成更大的产物。</p>

<p>酶的功能方式还有很多。例如，这种酶可将单一底物转变为新的构型；而另一种酶则可将一个底物的一部分转移到另一个底物上。有些酶无法独立催化反应，需要辅因子或辅酶才能催化其反应。</p>

<p>那么，你可能会问，这些内容与15世纪的航海有什么关系呢？当时，水手们无法获得含有维生素C的新鲜农产品，而维生素C是胶原蛋白合成所必需的辅酶。因此，水手们会患上坏血病——一种因缺乏维生素C而引起的疾病，且常常致命。他们通过腌制富含维生素C的水果和蔬菜来解决这一问题。腌制降低了食物的pH值，从而抑制了导致食物腐败的微生物酶的活性。因此，破坏微生物酶并保存这种必需辅酶，对长途航海的成功起到了关键作用。</p>

<p>在本实验中，您可以通过测定单位时间内产物的生成量（即反应速率），来定量分析不同条件对酶功能的影响。首先，您将在标准室温和中性pH条件下测定基础反应速率。反应初期，反应速率会逐渐增加，直至所有酶分子均被底物完全饱和；随后，由于酶持续进行底物结合、催化和产物释放的循环过程，催化反应将达到一个稳定水平。在确定基础反应速率后，可将其作为其他条件下反应速率的参照基准。本实验所使用的酶为辣根过氧化物酶（turnip peroxidase），您将评估该酶在不同温度和pH条件下的活性表现。</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

生物催化剂

所有生物体为了维持生命活动，会持续进行大量的生化反应。大多数这些反应需要……

使用 JoVE Lab Manual 上的这个Biology评估测验来测试你对酶活性的理解。通过视频问题涵盖关键概念，旨在评估学生的学习情况。

酶活性（Biology）- 评估测验 | JoVE Lab Manual

在这段 9 分钟的学生实验流程视频中，逐步学习 酶活性 的操作步骤。属于 JoVE Lab Manual 上的 Biology 系列。

Peroxidase Lab: pH and Temperature Effects

Learning Objectives

酶是什么？

酶如何催化反应？

什么是分解代谢和合成代谢反应？

Lab Prep

Transcript

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