Show Yourself, Asparaginase: An Enzymatic Reaction Explained through a Hands-On Interactive Activity.

Determining the catalytic activity of an enzyme can be the perfect method for its identification, for example during purification procedures or for isolation purposes. Herein, we used a pharmaceutically relevant protein to bring the concept of enzymatic activity to the classroom. We designed a hands-on interactive activity in which a medically relevant enzyme, asparaginase, was distinguished from a nonenzymatic protein based on its specific enzymatic activity. The experiment was carried out in the classroom, designed to impact different educational levels from elementary to high school. Our main purposes were to promote the emerging field of protein-based drugs as a source of scientific careers in bionanotechnology and to show the students an image of a "scientist" as that of a common and educated person working in an exciting profession. In addition of being inexpensive, this activity proved to be adaptable for various educational levels and can be easily implemented in different scenarios, for example, scientific fairs, some schools, and so forth.

Scientists usually share their research in scientific journals, conferences, symposia, and interviews. However, the need for understanding and public awareness of science has led the community to develop an outreach strategy enabling the education of different individuals regardless of their scientific background and to promote a new perspective of science. An educational activity is a useful tool to expose people to scientific instruments and methods, but in a daily life environment such as in schools, fairs, and so forth.

The term “a scientist in the classroom” can be used to summarize a common extension method that brings scientific practices into the school, to stimulate the interest and learning of science between students in a fun and familiar atmosphere.[1] In this work, we performed a hands-on interactive activity with elementary and high school students, to show them a recent application of biochemistry in the field of protein-based drugs. The activity was implemented during an outreach program which had two main objectives: to promote bionanotechnology as a scientific career, and to show an image of a “scientist” as a familiar role model for the students. The activity was very inexpensive and straightforward, so that it can be implemented in different scenarios, such as the classroom.

We chose proteins to be the theme for our activity as they are the prime topic of study in biochemistry. Proteins are the basic functional building blocks of living systems with a manifold of functions, including catalyzing metabolic reactions, signal reception and transduction, enabling mobility, regulation of genetic activity, and structural roles, among others. In recent years, many proteins have also been used to prevent and treat diseases, and recently, its medical applications have significantly increased. In the last three decades, more than 90 protein drugs entered to the pharmaceutical market.[2] Interestingly, it is possible to demonstrate, inside the classroom, the function of a protein that is currently being used in the treatment of cancer. This is beneficial to bring awareness among students on the existence of this emerging field (protein-based drugs) as part of the scientific concern for improving the quality of human life, specifically regarding cancer that has become a common issue in people’s social and cultural contexts.

Today’s educational standards at elementary and high school levels focus mostly on teaching the central dogma of biology (DNA → RNA → proteins), while much of the educational research at these levels focuses on enzyme kinetics.[3−6] Here we present proteins as essential materials in medical applications. Enzymes are proteins (and sometimes RNA molecules) that catalyze biological reactions; therefore, they accelerate chemical reactions occurring in living systems. The activity of an enzyme refers to its capacity to accelerate a specific chemical reaction(s) that without its intervention would proceed extremely slowly. In the laboratory, the presence of a certain enzyme can be screened within a mixture by measuring the specific enzymatic activity, even from a crude bacterial extract. After purification, the activity is a clear indicator that the enzyme remains in its functional state (active form).[7,8] Therefore, this is the preferred method used by biochemists to evaluate their enzyme of interest. Conveniently, some enzymatic reactions can be easily followed using a substrate that changes color upon transformation, which make them ideal for qualitative demonstrations.[9−11] In this work, we utilized an important protein drug to explain the concept of enzymatic activity and to show the usefulness of proteins in medical applications, by using a simple colorimetric reaction.

Theoretical Background

Asparaginase is an enzyme that catalyzes the hydrolysis of l-asparagine to l-aspartic acid and ammonia. This protein is a fundamental chemotherapeutic agent used in the treatment of acute lymphoblastic leukemia, in which the mechanism acts due to the inability of these cancer cells to synthesize their own endogenous l-asparagine (Figure ). By injecting asparaginase into the blood, it depletes the circulating concentration of l-asparagine until the malignant cells starve.[12]

Figure 1

Asparaginase chemotherapeutic action. Asparaginase catalyzes the hydrolysis of l-asparagine to l-aspartic acid. Since leukemia cells are unable to produce its own endogenous l-asparagine in a sufficient amount, the reduction of blood-circulating l-asparagine induces them to starve without affecting healthy cells.

In the laboratory, the enzymatic activity of asparaginase can be determined by the release of ammonia in coordination with Nessler’s reagent, which forms a yellow-to-orange product.[13,14] But this method is inappropriate for the classroom due to the presence of mercury in Nessler’s reagent. However, an alternative way to determine the presence of ammonia is by the salicylate method. Kits employing this chemistry are commercially available as aquarium test kits, because ammonia production in an aquarium can lead to fast and severe fish loss. In this method, ammonia (enzymatic product) reacts with hydrochlorine to form monochloramine, which then reacts with salicylate to form 5-aminosalicylate that exhibits an intense green color (Scheme ).[14]

Scheme 1

Mechanism for the Salicylate Method

Materials and Methods

Asparaginase from E. coli, bovine serum albumin (BSA), and l-asparagine were purchased from Sigma-Aldrich (St. Louis, MO). The ammonia aquarium test kit (API ammonia test kit, liquid formula) was purchased in a local pet shop and used as per the manufacturer’s instructions. The reagents bought were sufficient for about one thousand students, and we calculated the individual cost to be $0.40/student. Specific details regarding how to prepare the working solutions and carry on the asparaginase enzymatic reaction are found in the supporting files (Supporting Information 1A).

The Activity

Asparaginase was selected as the model protein for this activity because of its medical importance and its easy-to-identify enzymatic activity. Other protein drugs (as insulin) are not suitable for a hands-on activity as they have a more complex function. We believe that the theme of protein drugs was an excellent choice to fulfill our first objective, to promote bionanotechnology as a source of scientific careers, since it is a relatively new topic related to biochemistry, which combines knowledge from biology and chemistry, making the activity more attractive for a general audience. In addition, most of these proteins are safe to be manipulated outside the laboratory, like asparaginase in this case. Finally, asparaginase is among the few enzymes that are stable at room temperature and in water (as solvent), without losing its activity even after a prolonged storage period (Supporting Information 1B). Our second objective, to show to the students an image of a “scientist” as that of a common and educated person working in an exciting profession, was accomplished by using a graduate student to perform the demonstration. As an introduction, the student described some of his life experiences prior to entering graduate school and now working as a scientist (Supporting Information 2). This part of the activity was focused in showing the participants that being a scientist is not an ordinary profession and emphasizing that work and fun can be balanced. In the case of high school students, we spoke about career opportunities available in STEM areas.

This activity was designed to be implemented at different educational levels, from elementary to high school. To prove its possibilities, we performed the activity with students of the second and tenth grades. A total of 28 students from the second grade and 30 from the tenth grade participated. The activity consisted of a hands-on interactive experiment to show the enzymatic activity of asparaginase,[12,15] alternating with an animated slideshow to introduce basic concepts: bionanotechnology, protein size and composition, asparaginase as an antileukemia drug, recombinant protein production (in general), and the asparaginase enzymatic activity (Supporting Information 2). Finally, we finished the presentation with a competition between the two proteins (asparaginase vs BSA) (Figure ). Through this methodology, the students were motivated by the adaptation of a recreational chemistry strategy; they were entertained with an experiment in which scientific concepts were perceived as something else more than a “magical color change”.[16] More information regarding the methodology of the activity is found in the supporting files (Supporting Information 1C).

Figure 2

Asparaginase enzymatic activity. (A) Asparaginase presence. Asparaginase reacts with the substrate (l-asparagine) to form the enzyme–substrate complex and generate the product (ammonia); then the presence of ammonia is determined by the addition of the aquarium test kit solutions that shows a change of color (orange to green). (B) BSA presence. BSA is mixed with the substrate, and no reaction takes place; then the aquarium test kit solutions are added, and in this case the color remains (orange) since no ammonia is produced. Chemical structures are generated from published data[15] and visualized and modified with PyMOL and ChemDraw.

Students’ Response and Discussion

In the case of elementary school students, their attention focused mostly on the analogy of protein size and the animation of enzymatic activity. They were intrigued about the nanoscale size of proteins; questions such as “if proteins are so small, how we know they are there?” provided the perfect link to explain the concept of enzymatic activity and its usefulness in the laboratory. This type of visual animation methods, which simulate the interactions occurring between compounds in a chemical reaction, promotes interest among students and helps the understanding of scientific concepts at the molecular level.[17,18] It is important to emphasize that the students from the elementary school were more motivated and enthusiastic. They admired the experiment with expressions of excitement and celebrated every change in color.

High school students were less enthusiastic, but more inquisitive and specific in their questions. Many of them expressed great surprise by the fact that a simple enzyme could have such important medical applications and that they were able to interact with it in the classroom. We understand that, as the students advance to higher levels, they are less excited by this type of activity.[19−21] However, we believe that these activities expose them to applied fields of science and encourage them to follow a scientific career.[21]

This activity was interesting not only for the participants but also for the graduate students, since it obligated them to adapt their scientific knowledge to be understood by a general audience. This is a very important aspect, because most scientists have problems expressing their knowledge (scientific research) in a simple and practical way. Therefore, this kind of activity is a great opportunity to improve their communication skills.

Conclusions

Although during this activity we could not employ assessment methods, here our conclusions are presented in terms of the knowledge shown by the students, their enthusiasm during the activity, and the versatility to be implemented at different educational levels.

We noticed that most students already had a general knowledge about leukemia within a social context, even the elementary school students. On the other hand, almost all of them (including the high school students) did not know about the application of asparaginase in this treatment and had a vague concept of what chemotherapy is. As expected, elementary school students did not recognize the concept of enzymes, while most high school students had a general knowledge of this topic. Finally, only the high school students had previous knowledge of nanoscale size, but in a very theoretical sense.

We perceived that this activity generated enthusiasm and interest in science within both groups of students (elementary and high school); it was possible to teach scientific concepts in an entertaining way. We think that even the youngest students understood the concept of enzymatic activity, since they could distinguish between the presence of both proteins based on the final mixture color. To our understanding, this activity is adaptable for different educational levels, if the content and duration are modified, including a general adult audience. It can also be carried out by any person who possesses the basic knowledge here discussed, for example, a graduate student, teacher, and so forth, although we believe it is more enriching when it is done by a graduate student.