Authors: GEORGIOS CHATZIGEORGIOU, biology teacher, 2nd General Lyceum of Oreokastro, Greece, ALEXANDROS TERZOPOULOS, Mentor

We would like to express our utmost gratitude to Ms Konstantina Sarantavga, Principal of the 2nd General Lyceum of Oraiokastro, for her continuous help and support in implementing the STEM activity in our school.

General overview of enzymes

Enzymes are proteins capable of catalysing biochemical reactions in living organisms. As biocatalysts, they accelerate such reactions by lowering the activation energy thereof. Enzymes possess a varying degree of specialisation with respect to the reactants (substrates) of the reactions they catalyse; for their action, the binding of the substrate to the region of the protein known as the active site is required (in a lock-and-key manner). The velocity of enzymatic reactions is affected by temperature, substrate and enzyme concentration, binding affinity, pH etc. The enzyme function may be halted or increased by the presence of various substances (e.g. inhibitors, activators, allosteric effectors). Inhibition may be reversible or non-reversible.

Relevance to school curriculum-Benefits of our approach

The subject and encompassed disciplines are part of the curriculum for the General Orientation Biology course mandatorily taught in the 2^nd Class of the General Lyceum (5^th year of secondary education) in Greece. Enzymes specifically are taught within the framework of Chapter 3 “Metabolism”, section “Enzyme-Biocatalysts”. The time allocated for this section is one class sessions for the theoretical part, and an additional 1-2 hours to complete the experimental section designed herein.

The teaching approach we followed replaces the typical “dry” theory-repetitive presentation of this subject in the class with inquiry-based learning, including a hands-on experience in the lab with a strong visual stimulus. In our first implementation, 15 students participated (most of them girls) who showed great interest in the way the STEM fields of Biology and Chemistry were explored with this activity.

The utilisation of an interactive approach, especially one involving laboratory work, have been noted to generate interest among pupils, while also allowing the educator to organise and adjust the activity via a constant feedback loop with students’ experience. Notable is the intuitive presentation of specific aspects of a subject not approachable by other, traditional means. 

Most of a pupil’s progress in school-taught STEM courses – such as Biology, Chemistry, and Physics – is a result of critical processing of the subject material viewed also through the lens of previously acquired knowledge; such knowledge is very effectively mastered in an environment which harbours conversation and exchange of ideas. To this end, the teacher may utilise various multimedia such as slides, educational videos, computer simulations, solid molecular models, practical demonstrations, lab experiments and more. The content thereof is always connected to the official curriculum and textbook, but in this manner the student can be encouraged for further discourse and more active participation. Concurrently, the students exercise in organising scientifically relevant arguments to support what they observe and conclude therefrom, as well as modifying those when presented with additional facts.

A laboratory exercise is particularly suited for the aforementioned as it does not keep pupils passive receptacles of textbook knowledge but rather engages them in what truly is the scientific method: empirical inquiry of physical phenomena in a controlled environment where the only objective truth is no other than the outcome of the experiment. Thusly, the student can follow the steps which are used in the Natural Sciences to discover in the first place what they are otherwise taught as necessary true within a textbook. This also contributes to the teaching of the subject as a whole, cultivating desirable skills such as scientific observation, accurate measurements, classification of phenomena, proposing a hypothesis, reaching empirical conclusions, and comparing these to known results.

Practical implementation

For this IBSL activity we specifically chose the section “Enzymes-Biocatalysts”, judging it to be fertile ground for combining (a) general educational goals such as the development of critical thinking or cooperation on all levels of teaching, and (b) targets specific to Natural Science and STEM subjects such as the empirical method of inquiry and the design of an experimental setup.

In our scenario, the activity is implemented in two distinct sessions of approximately 1 hour’s duration each. For the first session, teams of 3-4 students each participate actively in worksheet-related exercises requiring the use of the Internet for research as well as a dialogue between teammates, cataloguing of their conclusions and presentation to the rest of the class. The educator acts as a guide, contributing to the completion of the assignments while concurrently moderating the presentations and constructively assessing each team’s results.

During the second session, each team undertakes the design and implementation of an experimental setup, catalogues their hypotheses, and carry out a series of experiments to collect empirical data confirming or disproving the formulated predictions.

Materials/equipment needed for implementation

To complete the 1^st worksheet, students (in teams of 3-4 individuals) need to use the school computer room in which computers have to be equipped beforehand with folders containing the selected educational material and appropriate software (animations and videos related to the topic -all in the native Greek language of the students- and the necessary programs for multimedia playback, text/image editing, PowerPoint-type presentation software). Alternatively, the session could be run in the school science lab as the second session provided appropriate projectors and portable computers are available.

For the 2^nd worksheet, the experimental exercise needs be carried out in a school science lab as mentioned; the lab space should be able to accommodate the 3-4 student teams. Extremely specialised/dedicated equipment is not required as the apparatus we built can be replicated with common (or easily obtained) laboratory instruments and materials (beakers, test tubes, volumetric cylinder, plastic tubing, metallic tightener, a three-way valve; presented analytically further below). As for reagents, hydrogen peroxide solution can be very easily obtained as a commercial 3 % solution sufficient for the purposes of the experiment, along with fresh cow’s kidney, and standard saline solution.

Outline of the 1^st worksheet

This is completed first during the preparatory session before the experiment; it consists of 2 in-class activities encompassing 6 questions, a concluding question, and a team project as homework. For their completion, the teacher acts as a mentor and tries to achieve all the aforementioned educational goals.

1st WORKSHEET: Enzymes’ Mechanism of Action

1st activity

Think: what do molecules require in order to successfully react with other molecules? E.g. how may gaseous nitrogen and oxygen react in the atmosphere?

Task A: Discuss with your team and write down your thoughts in the space below

………………………………………………………………………………………………………………………………………………

(all teams present their opinions in front of the class)

Evaluate the validity of your assumptions after completing the task below.

Task B: Watch the related videos “Activation energy” and “Catalysts and Activation Energy”. Then answer the following questions:

B1: What is the requirement for molecular bonds to break?

………………………………………………………………………………………………………………………………………………

B2: How do you comprehend the concept of activation energy? How is this barrier overcome?

………………………………………………………………………………………………………………………………………………

2nd activity

Think: How do chemical reactions proceed inside the cells of a living organism? How is the energy barrier overcome in this case?

Task C: Discuss with your team and write down your thoughts in the space below

………………………………………………………………………………………………………………………………………………

(all teams present their opinions in front of the class)

Evaluate the validity of your assumptions after completing the task below.

Task D: Watch the related video “Activation energy 2”. Then answer the following questions regarding enzymatic function:

D1: Sometimes chemical reactions don’t proceed rapidly enough under standard equations because

they have a low activation energy

they have a high activation energy

the products are not sufficiently stable

they are endothermic

they are exothermic

D2: In an enzyme-catalysed reaction what changes is

the reactants

the products

the activation energy

the enthalpy

all of the above

Task E: Watch the related video “How Enzymes Work”. Then answer the following questions based on the scheme below:

E1: Identify the enzyme, substrate, and products

E2: Name stage A

Task F: Watch the related animations “Enzymes” and “Enzyme Catalysis“. Then answer the following questions regarding enzymatic function:

F1: In the absence of the enzyme the substrates

a. react slower to give products

b. react faster to give products

c. react with a different stoichiometric ratio

d. do not react at all

F2: In a chemical reaction the enzyme

a. is consumed stoichiometrically

b. decreases the reaction velocity

c. is regenerated upon reaction completion

d. is one of the reactants

e. is one of the products

F3: Why do enzyme and substrate possess a complimentary structural relation?

a. So that substrate binding is easier

b. So that substrate bonds are broken more easily

c. So that the enzyme possesses high specificity

d. All of the above

Teamwork Task for home: Each team should try to list as many products as possible, that we consume daily and contain enzymes. Explain what is the role of enzyme presence in these products.

Outline of the 2^nd worksheet

This is completed next during the preparatory session before the experiment; it consists of 8 open-type questions meant to prepare students for the experimental session. The teacher’s guidance is strongly advised in this section as the material is much more specialised and students ought to present credible sources when conducting their research.

2nd WORKSHEET: Focus on Peroxidases

A general introduction to peroxidases Peroxidases are a large group of enzymes of the reductase family; they participate in a large number of reactions, many involving the breakdown of peroxides (hence the name). A large number of these is produces in the mammalian kidney; in next session’s experiment we will see how kidney tissue can break down hydrogen peroxide (H2O2) into water and gaseous oxygen which evolves as bubbles in solution. An unbalanced reaction is given:

After conducting your research as teams and citing credible sources, answer the following questions:

Question 1: Which peroxidases are usually found in kidneys?

………………………………………………………………………………………………………………………………………………

Question 2: Why is their concentration so high in this tissue?

………………………………………………………………………………………………………………………………………………

Question 3: What is the stoichiometry of the reaction?

………………………………………………………………………………………………………………………………………………

Question 4: Which gas evolves during the experiment? How can we measure the volume thereof accurately?

………………………………………………………………………………………………………………………………………………

Question 5: How could the phaenomenon of active site inhibition be observed experimentally?

………………………………………………………………………………………………………………………………………………

Question 6: Are there any safety considerations for the above experiment?

………………………………………………………………………………………………………………………………………………

Question 7: What is the importance of the breakdown of hydrogen peroxide for human health?

………………………………………………………………………………………………………………………………………………

Question 8: How do you judge the role of peroxidases for homeostasis?

………………………………………………………………………………………………………………………………………………

Experimental setup and activity            

A particularly visually appealing and effective way we chose to quantitatively measure enzymatic action is by studying the disproportionation reaction of hydrogen peroxide in the presence of peroxidases; this reaction evolves oxygen gas as one of the main products and thus offers a very direct and quantifiable way of visualising the effect of the enzyme. The concept of studying gas-evolving reactions can be quite useful in the study of STEM subjects already from the start of secondary education, especially from the 4^th year onwards (esp. after methods such as stoichiometric analysis are introduced to students). However, carrying out a homogeneous reaction with gaseous products is usually prohibitive in a school lab due to the time required or the inaccuracy of crude volume measurement methods available in this environment

Thusly, the construction and study of an experimental apparatus (based on the work of Giannakoudakis-Parisopoulou) capable of collecting and measuring to a satisfying accuracy the produced gas is very useful in enabling the completion of robust, fast, and reproducible experiments in the school lab. The results of such experiments need not be confined to the study of gas-evolving enzymatic reactions as we chose herein, since they can be generalised for multiple chemical/biochemical phaenomena of interest to secondary education instructors being an invaluable tool as discussed previously. The specifics of the apparatus construction are presented in the following paragraphs.

The entire setup uses easily obtainable parts, and consists of the three main functional elements described here (see Fig. 1 for an annotated photograph of the final apparatus built):

• Gas Collector (C in Fig. 1)

A transparent plastic graduated cylinder, available in any standard equipped school lab, of 500 ml lab and 2 ml marks was used as the gas collector. Along its bottom side was opened a hole with a drill and a small plastic tube of appropriate diameter was glued with a silicon gun. The tube has to fit tightly with the propipetter (P in Fig. 1) which generates the vacuum needed to raise the liquid level up to the top of the inverted cylinder.

The collector is held in place with the help of a clamp, and inside it is placed the tube T1 which leads the gaseous product from the reaction vessel into the collector (where the displacement of liquid from the initially filled inverted cylinder provides a rather accurate gas volume measurement). A metallic tightener (S) is also present so that communication between the vessels can be interrupted when appropriate (e.g. when initially filling up the collector with the propipetter).

• Reaction vessel (RV)

As the reaction vessel was used a transparent plastic milk bottle; this has the great advantage (in addition to minimal cost and availability) that it possesses an airtight seal, preventing gas leak. Two holes are easily bored into the plastic cap, wherein the two plastic tubes can be glued; one (T1) is for transferring the produced gas to the gas collector, while the other (T2) adds initially the liquid reagent (herein hydrogen peroxide solution) from the syringe into the reaction vessel.

• Syringe (Sy)

The 50 ml syringe (with 1 ml graduation) is utilised for feeding the reaction vessel with an accurately measured volume of the liquid reagent of choice. By setting the three-way valve (SC) to the appropriate position, the reagent is led from its container (e.g. a beaker, simply denoted H­₂O₂ in Fig. 1) to the syringe; it may be then disposed to the reaction vessel through tube T2 by setting once more the valve in the correct configuration. The syringe once emptied may then be rapidly filled with a new volume of reagent while pneumatically isolated from the rest of the system, adding to the versatility in the system’s operation.

After building the apparatus as described in the school Chemistry lab of the 2^nd General Lyceum of Oraiokastro, the experiment was run by students during the second session of this IBSL activity. Regarding technical details, the hydrogen peroxide source used was a low-cost commercial antiseptic of 3 % concentration, posing minimal hazard compared to more concentrated versions of the reagent used in other kinetics experiments.

As for the peroxidase source, animal kidney tissue from a local shop was used (stored under refrigeration until before the experiment); kidneys of cow, goat, and lamb were tested for relative comparison (the cow’s one giving the biggest response). Before each run, the kidneys were carefully washed with saline solution (NaCl 0,9% w/v) and cut into samples of precisely weighed mass (65 g). Each sample was pulverised and an appropriate volume of saline solution (about 25 ml) was added to each, as to reduce the viscosity of the tissue sample while retaining a constant osmotic pressure. The sample was then added to the reaction vessel and the apparatus operated by the method described above (initially the collector is filled to the top with liquid, the syringe with reagent which is then added, valves appropriately closed, and all the gas directed to the gas collector for accurate volume measurement).

Ideally each experiment is to be repeated 12 times (4 samples from each animal) by each team, but usually time constraints make this unfeasible. Instead, each team can be assigned two animals tissues to test fully after familiarising themselves with the operation of the apparatus, and then results such as average values and standard deviation can be compared across the class. The students could be asked to interpret the results (a follow-up from the 2^nd worksheet), as well as present the insight gained from the previous session by answering relevant questions during the course of the experiment.

Final section: Questions for assessment and feedback of the activity

A.  Feedback from the educator

Have the initially set educational goals been met?

………………………………………………………………………………………………………………………………………………

Which additional educational goals may be achieved?

………………………………………………………………………………………………………………………………………………

Which activity do you think was most effective at achieving the desired educational goals?

………………………………………………………………………………………………………………………………………………

Have students freely expressed their opinions during class discussions?

………………………………………………………………………………………………………………………………………………

How has the use of videos/animations affected students’ learning?

………………………………………………………………………………………………………………………………………………

Do all students participate equally in activities? Is teamwork carried out in a balanced manner from all participant?

………………………………………………………………………………………………………………………………………………

Suggest ways to alter the educational activity as to enrich its scope with further topics of a similar level and which can be approached by a similar technique.

………………………………………………………………………………………………………………………………………………

B.  Feedback from the student

With regards to teamwork

Did each member of the team take on a specific subsection of the assignment?

………………………………………………………………………………………………………………………………………………

Did you work effectively as a team?

………………………………………………………………………………………………………………………………………………

Did you discuss the purpose of each activity/task?

………………………………………………………………………………………………………………………………………………

Additional comments/issues/suggestions

………………………………………………………………………………………………………………………………………………

With regards to the worksheets

Were the worksheet instructions clear? Did you follow them exactly?

………………………………………………………………………………………………………………………………………………

Were the videos and animations understandable? Helpful?

………………………………………………………………………………………………………………………………………………

Additional comments/issues/suggestions

………………………………………………………………………………………………………………………………………………

With regards to the topic

Have you understood enzymes’ mechanism of action and their properties?

………………………………………………………………………………………………………………………………………………

Is there any part of the topic which was not analysed sufficiently? Anything you would have liked to be more of?

………………………………………………………………………………………………………………………………………………

Additional comments/issues/suggestions

………………………………………………………………………………………………………………………………………………

With regards to the experiment

Have you understood what was illustrated by the experiment?

………………………………………………………………………………………………………………………………………………

How much has this helped you contextualise the theoretical topic?

………………………………………………………………………………………………………………………………………………

Have you followed the experimental procedure accurately? Can you account for every step in the procedure?

………………………………………………………………………………………………………………………………………………

Can you account for the controls and assumptions of this experiment?

………………………………………………………………………………………………………………………………………………

Additional comments/issues/suggestions

………………………………………………………………………………………………………………………………………………

• 

• 

Participating students: 

B3 class: Dimitrios  Aspridis, Andriana – Pandora Vasileiou, Dimitra Gianniou, Maria Varangouli, Kapetanios Vasileios, Maria Karatza, Chrysa Kordela, Katerina Koutsokosta, Dimitra Kapsali, Kyropoulou Stavroula, Vasileios Markopoulos, Kourkouta Anna, Katsareli Katerina.

A5 class: Anastasia Takatini, Schina Vasiliki, Chara Tzelepi, Kelly Pelteki,  Alexandra Rafailidou, Efthimia Stefanidou.