Enzymes are proteins that function as biological catalysts, which increase the rate of reaction without being destroyed themselves by it (Campbell, et al., 2008). Since not all chemical reactions are fast acting, the slower reactions need a “boost” and that is where enzymes come in. A major process that occurs within our bodies is metabolic process, which tends to be a slower reaction. If metabolic process were to occur at its normal slow rate than many cells would die. Thus, the vital role of enzymes is key for slow acting processes naturally occurring in our bodies.
Enzymes are substrate-specific, meaning that only a particular substrate can react with the enzyme to make a product. The given substrate fits into a region of the enzyme called the active site. The active sites act like a “pocket” for the substrate. In this particular lab the enzyme being used was tyrosinase, which is found in many organisms including humans.
All enzymes have an active site, and an enzyme is “available” when the active site is empty. Then comes the substrate, which binds to the enzyme in the active site. With some enzyme-substrates hydrolysis occurs, but it may not always be the case. After, the substrate is converted into products. Then, the products are released. There is a huge turnover count for products because this cycle occurs in the thousands range per second.
The three main environmental factors that affect enzyme activity are temperature, pH, and substrate concentration. Temperature and pH are components of homeostasis, which help keep our bodies’ cells in equilibrium. As the bodies temperature increases so does the enzyme activity. Then at about 37° Celsius enzyme activity reaches its optimum, which also happens to be the bodies average temperature.
After the optimum point, the enzyme activity begins to drastically decrease. Enzymes tend to be hypersensitive to high temperatures, because it is at this point that they tend to denature, or “unwind.” The two main things that cans cause the denaturing of enzymes are the severity and duration of the heat. Enzymes can be renatured if the temperature decreases back to body temperature.
Tyrosinase is a naturally occurring enzyme in plant cells. This particular enzyme causes the brownish coloring from bruises in fruits and vegetables. At the site of “injury” tyrosinase reacts with pyrocatechol (a substrate) to then form hydroxyquinone. In this particular lab, the tyrosinase comes from potato cells (Couch and Beger, 2004).
The control experiment tested four different combinations of enzymes and substrates. The first was the enzyme tyrosinase and the substrate pyrocatechol and the second was just the substrate pyrocatechol. The third test tube had just the enzyme tyrosinase, and the last test tube had the enzyme tyrosinase and the substrate sucrose. Temperature was the experiment being tested by the group. There were four environmental temperatures in which the enzymes were exposed to, ranging from cold to hot water baths.
After discussing the role of enzymes we were able to synthesize a class hypothesis for the control experiment, which was that a specific enzyme and substrate bind to create products through a catalyzed reaction. The hypothesis was produced with the knowledge that certain substrates are specific to enzymes and if the combination is not “right” than there will be no reaction. The two predictions we were able to make for the hypothesis were that tyrosinase and pyrocatechol will create hydroxyquinone in the first test tube, and that there will be no reaction if tyrosinase and pyrocatechol are not combined.
The second experiment, which tested the effect of temperature on reaction rate and enzyme activity, was the assigned one for my particular lab group. The hypothesis for this experiment was that the temperature of the environment determines the rate of change in a catalyzed reaction. We were able to create our hypothesis because we knew that enzymes are catalysts and are able to help reactions occur in colder than normal temperatures and also have an effect in temperatures deemed “normal” but may not have a positive outcome in warm temperatures because of the “added” heat. The first of the three predictions was that the test tube in the refrigerator will produce a positive reaction, therefore will change from its initial coloring. The second prediction was that the test tube at room temperature will produce a positive reaction, therefore will change from its initial coloring. The last prediction was that the test tubes in the hot water bath will not produce a positive reaction, therefore will remain the initial color.
The controlled experiment had four test tubes and two results, either a negative or positive for hydroxyquinone. The initial color for the first test tube was a clear color with a slight cloudiness, after five minutes the contents’ final color was a clear dark yellow; this first test tube is the positive control for the experiment. The second test tube was initially clear with the final color having a slight cloudiness. The third test tube, which solely contained the enzyme initially, had a clear with slight cloudiness color, with the final appearance being the exact same. The last test tube had a different substrate, sucrose, with the initial appearance being clear with cloudiness and the final having a pale pink with a slight cloudiness. The last three test tubes are negative controls for the experiment. A reference to Table 1 can further illustrate the test tubes and their colors.
In the temperature experiment there were four results as well. The test tubes were placed in four temporal environments. The first of which was the “cold” one, which was placed in the refrigerator at 3°C for approximately fifteen minutes before introducing the enzyme and substrate. The initial color for this tube was clear without the enzyme or substrate, and remained clear with the substances. After five minutes of being returned to the original temperature the final color remained to be clear. The second test tube, room temperature, was at 21°C; the initial color without enzyme or substrate was clear, and with them it turned into a cloudy with light yellow tint. After the last five minutes, the final color of the contents was a golden translucent.
The last two test tubes were placed in the hot-water bath at two different temperatures. The first warm temperature was at 26.8° C, without the enzyme and substrate it was a clear color, and with them it turned into pale yellow ting. The final color for this tube was a golden, dark color. The second warm temperature was at 34.9° C; whose initial color was clear, then with the enzyme and substrate it turned into a clear cloudy color. The final color was a pale pink translucent color.
The class-generated hypothesis for the control experiment was that a specific enzyme and substrate bind to create products through a catalyzed reaction. This is rather reasonable given the fact that certain enzymes are substrate specific as stated in the Introduction. With this specific experiment the enzyme tyrosinase and the substrate pyrocatechol were the main focus. Therefore, it would be safe to infer that if these two should combine, it will provide a positive result for hydroxyquinone. The lab manual also went into a brief over view as to what the purpose of each of these is separately as well as when they are combined. If there is a positive result for hydroxyquinone then the contents of the given test tube would turn into a yellow-brown; tyrosinase on its own is a brown color and pyrocatechol on its own is a clear color.
With this given information the group was able to make a hypothesis of how the temperature would affect the enzymes activity. The hypothesis was a bit vague because of the “extreme” temperatures that the enzymes-substrate mixtures would be exposed to. The hypothesis was that the temperature of the environment determines the rate of change in a catalyzed reaction. We knew that as long as tyrosinase and pyrocatechol were together there should be a reaction of some kind, but then when we added the temperature factor we also knew that not all the temperatures would have a positive reaction, and that possibly the amount of time that would be given (approximately five minutes) would not suffice for a ruction to occur.
In the controlled experiment the positive control was the first test tube, which contained the enzyme tyrosinase and the substrate pyrocatechol. This was visually proved when the test was conducted and the end product had turned into a clear, dark yellow tint. Which also showed that the substrate would produce hydroxyquinone when catalyzed with tyrosinase. The remaining three test tubes for the controlled experiment all were negative for hydroxyquinone. This is due to the fact that tyrosinase needs pyrocatechol to produce hydroxyquinone, and vice versa. The last test tube which contained the enzyme tyrosinase and the substrate sucrose had no reaction because when these two are combined they no reaction which will result in a positive result for hydroxyquinone.
In the actual experiment testing for the effect of temperature on enzyme reactions the results were split. There were three main temperatures, in which one of them had a double with the different temperatures. The first test was for a refrigerator temperature reaction, and the final color was clear, which is nowhere near a yellow-brown color, so the cold temperature was a complete negative for hydroxyquinone. This may have to do with the fact that the temperature may have been too cold and the enzyme may have denatured, but if it were to have increased to a higher sustainable temperature it would have most likely renatured itself.
The second test tube was left at room temperature, and the final resulting color was a golden translucent color, thus indicating a positive result for hydroxyquinone. This is due to the fact that enzymes are at their optimal level when at a “right” temperature, be it room or body temperature depending on the particular enzyme. Because potatoes, as well as most fruits and vegetables, are usually stored at room temperature their optimal level would be room temperature.
The last two test tubes were in hot-water baths, one was at 26.8°C and the other at 34.9°C. The test tube which was at 26.8°C had a final color of golden dark, which is a clear indicator for hydroxyquinone, therefore testing positive. It is also the only temperature that is remotely close to the room temperature of 21°C. The second test tube which was measured at 34.9°C and had a final color of pale pink translucency had a negative result for hydroxyquinone.
For a greater majority of the experiment the hypothesis and predictions were right on target for the controlled and actual experiment. The only part that showed inconsistencies was on the temperature experiment in the refrigerator and hot-water bath test tubes. As a group we predicted that the refrigerator temperature would result in a positive when in actuality it was a negative result which may have to do with the fact that the temperature was far too cold for the enzyme and therefore resulted in it denaturing. As for the hot-water baths we had predicted that
they would both result in a negative reaction, when in fact only one of them had a negative result and that one happened to be the one that was the warmest out of the two. This may have to do with that one denaturing as well, just the same as the refrigerator temperature one.
The experiment was a bit limited to just these four temperatures, also possibly if the test tubes would have been allowed more than five minutes in each of its respectable environmental temperatures than possibly some of the results may have differed a bit. As well as not just limiting the experiment to tyrosinase, pyrocatechol, and sucrose. If there would have been a bit more variety in enzymes and substrates it would have been interesting to see the results for each of the differing combinations.
On the matter of hydroquinone’s scientific and societal benefits, in ScienBlogs it was mentioned that hydroquinone is an antioxidant, but has signs of toxicity and a potential carcinogenicity, meaning a potential for cancer. This is rather interesting, in particular because hydroxyquinone is easily made on fruits and vegetables when they have become bruised. All in all the experiment was a bit beneficial to us, we now know that this catalyzed reaction can be seen on fruits and vegetables, as well as that they may be lethal, depending of course the amount and many other factors that may influence its use.
–2008. “Hydroquinone (Antioxidant or Toxin).” ScienceBlogs. Retrieved 3-20-10 from
Campbell, N. A., Reece, J. B., Taylor, M. R., Simon, E. J., and Dickey J. L. 2008. In Biology Concepts and Connections 6th Ed, pp. 84-85. San Francisco. Benjamin Cummings.
Couch, L., and Berger, L. R. 2004. Enzymes: A Qualitative Approach. In Biology: Lab Manual for Biology 124. 5th ed. Boston: Pearson.
Table 1: Controlled Experiment
|Test Tube||Test Tube Contents||Initial Color||Final Color||Result|
|1||Enzyme (tyrosinase) & Substrate (pyrocatechol)||clear with slight cloudiness||clear dark yellow hue||Positive|
|2||Substrate Only (pyrocatechol)||clear||clear with slight cloudiness||Negative|
|3||Enzyme Only (tyrosinase)||clear with slight cloudiness||clear with slight cloudiness||Negative|
|4||Enzyme & Substrate (sucrose)||clear with cloudiness||pale pink with slight cloudiness||Negative|
Table 2: The Effect of Temperature On Reaction Rate/ Enzyme Activity
|Initial Color||Final Color|
|Test Tube||Tube Placement & Temp||Without Enzyme||With Enzyme & Substrate||After 5 Mins||Result|
|1||Refrigerator (3°C)||Clear||Clear||Pale/ Translucent clear||Negative|
|2||Room Temp. (21°C)||Clear||Cloudy with light yellow tint||Golden translucent||Positive|
|3||Hot H2O Bath (26.8° C)||Clear||Cloudy clear||Pale pink/ Translucent||Positive|
|4||Hot H2O Bath (34.9° C)||Clear||Pale yellow tint||Golden/ Dark||Negative|