The Effects of Catechol Concentrations and Temperature on Benzoquinone Production - Lab Report Example

The Effects of Catechol Concentrations and Temperature on Benzoquinone Production

Timothy S. O’Neal

Biology Lab 2051

Dr. Celeste Lajala

18 October 2017

The Effects of Catechol Concentrations and Temperature on Benzoquinone Production

There are millions of chemical reactions taking place in our bodies every second of every day. A chemical reaction of the utmost importance is the work of enzymes. Enzymes are molecular reactants that help kick start, or synthesize chemical reactions within a cell. Without enzymes our cellular processes would start, and move along much too slowly to support life. The definition of an enzyme is, any of various complex proteins produced by living cells that bring about or speed up reactions (as in the digestion of food) without being permanently altered (Merriam-Webster's collegiate dictionary, 1999). The human body’s ability to break down sugar and turn it into energy is a good example of this important process. There are six different types of enzymes found in our bodies. The enzyme hydrolases is one that helps us digest our food, and is involved in food decomposition and in the digestive process. These enzymes help us break down proteins and carbohydrates to use as an energy source. This paper’s focus is to make predictions on the effects that physical factors, such as temperature, will have on the rate of enzyme reactions.

The current experiment majorly focused on catecholase enzyme which is usually present in fruits and vegetables. It is involved in the facilitation of product browning after being exposed to oxygen. This catecholase enzyme was generated from potato. The observed browning reaction was facilitated by the oxygen molecule and the catechol and in this case, benzoquinone was considered to be the browning product. It is well known that the majority of enzymes in humans work optimally between 35 and 40 degrees Celsius which are considered to be normal body temperature. The experiment’s main objective was to determine the impact of substrate concentration (catechol) and temperature on catecholase (or benzoquinone production). Based on the fact that the temperature of human body is relatively constant, it can be hypothesized that the enzymes in the consumed food react optimally at about 37 degrees Celsius. The testing of this hypothesis was through the catecholase activity which was observed at five temperatures of the substrate which ranged between 0-120 degrees Celsius. Based on the fact that temperature increase will lead to increase in the collision levels between the substrate and the enzyme, it is expected that the reaction rate will increase. In cases where the temperatures are relatively high, the enzyme is expected to change in structure such as being denatured. This means the reaction cannot be catalyzed by the enzyme as a result.

Substrate concentration was another variable studied in the current experiment. The substrate concentration was increased in different levels (0.2, 0.5, 1, 2.5 & 3 mL) to evaluate enzyme activity. Based upon this, a hypothesis was formulated as: the reaction rate increased with increase in the substrate concentration. The increase in the concentration of the substrate was maximal at the time where the substrate has occupied all the enzymes. The formulated hypothesis reflects the predictions of the experiment.

The reaction observed in the current experiment entailed catechol conversion to benzoquinone and thus it was facilitated by polyphenol oxidase enzyme (Creveling, 2000). The experiment results were used in undertaking different analyses accordingly. The catechol color changed in the course of the experiment.

Enzymes have an optimal operating temperature range that allows them to function at their highest level. The temperature of the human body () is the perfect range for these enzymes to operate at maximum efficiency. At higher temperatures the enzymes effectiveness decreases and they begin to break down. The current experiment was designed to measure the factors that cause an enzymes catalyzed reaction. The enzyme catecholase was used for this purpose, with the desire to create the end product benzoquinone. The catecholase was used as a catalyst to create a reaction in the substrate turning it to benzoquinone which is reddish brown in color. This change enabled the determination when the colorless substrate had changed into Benzoquinone.

Methods

This section explains how the two experiments were undertaken and in this case they were based on the effect of temperature and substrate concentration on the production of benzoquinone. Thus, the main objective was to determine the impact of substrate concentration (catechol) and temperature on catecholase (or benzoquinone production). In addition, the two experiments aimed at predicting the expected outcome where it was expected that the catecholase works optimally at 37oC. This was evaluated based on the temperature range of 0oC to 100oC. On the other hand, to determine how substrate concentration affected benzoquinone production, different concentrations of samples of the substrate were considered. This was evaluated against absorbance as evident in the subsequent sections. The method section was therefore divided into two subsections and they included temperature and substrate concentration.

Temperature

Benzoquinone production rate measurement in the presence of the catecholase enzyme was achieved by using a spectrophotometer. In this case, the solution absorbance was read in the spectrophotometer where the wavelength was set at 540nm. A higher concentration of benzoquinone resulted to a darker solution and hence a higher absorbance. Thus, the measurement of reaction rate was by absorbance increase with time. Solanum tuberosm (mashed potato) was used in the preparation of catecholase where 20 grams of it was put in 500 ml of water. A grease pencil was used to label 3 tubes as 1, 2, and 3. Labeling of the 3 pipettes was undertaken as catechol, potato and water and this was done to avoid cross contamination during the process of measuring. The first step was making of the blank which consisted of 9ml of distilled water and 3ml of potato extract (they were put in tube 1 after measurement) (Table 1). Secondly, the measured catechol (4ml) was put in tube 2(Table1). The third step entailed measuring distilled water (5ml at 25oC) and extract of the potato (3 ml) in tube 3(Table 1). The spectrometer was zeroed out using part of the solution of tube 1 where it was placed in a cuvette. Later, the cuvette was emptied and then rinsed using distilled water before drying it upside down on a towel. The reagents in both tube 1 and 2 were mixed for 15 seconds and then put in cuvette and spectrometer respectively. The absorbance readings were recorded at an interval of 20 seconds.

The second, third, fourth and fifth experiments were undertaken by first making blank reagent which consisted of 9 ml of water and 3ml of potato extract (it was put in tube1).The subsequent steps were same as the ones in experiment 1 above where different temperatures were considered(0,50,100 and 120 degrees Celsius).

Table 1. Summary of different Volumes of the Reagents used for Temperature Experiment

Tube

Distilled H2O (mL)

Catechol(mL)

Extract of Potato(mL)

1

9

3

2

4

3

5

3

Substrate Concentration

This experiment required different apparatus and chemicals and they included spectrophotometer, timer, 100mL beakers (2), pipet (1, 10 and 5mL), test tubes, 6.0 pH)phosphate buffer,0.1 percent substrate (catechol) and extract of potato with polyphenol-oxidase enzyme. There was preparation of 6 catechol samples with different concentrations as evident in Table 2.

Table 2. Summary of Volumes of used for Substrate Concentration Experiment

Sample

Substrate (mL)

Buffer (mL)

Enzyme (mL)

Volume(total-mL)

1

0

4

1

5

2

0.2

3.7

1

5

3

0.5

3.4

1

5

4

1

3

1

5

5

2

1.5

1

5

6

2.5

1

1

5

To ensure accurate measurement, the extract of potato was added the last in the mixture where the time was recorded instantly. The absorbance readings for the tubes were noted down at an interval of 20 minutes.

Results

The result section presents the results for the experiment that entailed the effect of substrate concentration and temperature change on the enzyme catalyzed reaction to generate benzoquinone. The summarized data and the analyzed data (presented in graph form had a certain trend which was compared to the expected outcome or formulated hypotheses. The generated data was analyzed and presented in form of graphs as seen in the next section.

Temperature

The temperatures with corresponding absorbance readings were recorded and presented as seen in Table 3. Four temperatures with corresponding absorbance were recorded and analyzed.

Table 3. Temperature with corresponding Absorbance Readings

Temperature (Celsius)

Absorbance

0

0.238

25

0.15

50

0.46

100

0.067

Based upon the results, it is evident that the highest absorbance was at 50oC (Figure 1). There were 5 temperatures considered in the experiment and they included 0, 25, 50, 100 and 120. In this case as observed in figure 1, there was a least absorbance at temperature 100 and 120oC.This was followed by 0 and 25oC where 50oC had the highest absorbance. The reaction rate was the highest at around 50oC and the least at 100 and 120oC.

Figure 1. Absorbance versus Temperature

Substrate Concentration

In order to determine how substrate concentration affects the production of benzoquinone, data on catechol concentration and benzoquinone production rate was generated. In this case, data for benzoquinone production rate at different substrate concentration is presented in Table 4. The benzoquinone production rate was recorded with corresponding sample and its concentration. This data was later utilized in generating a graph for benzoquinone production rate versus substrate concentration as seen in Figure 2.

Table 4. Data for Benzoquinone Production Rate and Substrate Concentration

Sample

Concentration of catechol(substrate)

benzoquinone production rate

1

0

-0.005

2

0.3

0.035

3

0.6

0

4

1

0.014

5

2.5

0.074

6

3

0.092

Benzoquinone production rate at different substrate concentration at a range of 20 minutes was presented as shown in Figure 2. In this case, the enzyme reaction kinetics was measured by benzoquinone production rate through the observed values of absorbance of the 6 different catechol concentrations. The production rate was seen to increase with increase in the concentration of the substrate. The equation of the curve is observed to be: and with . It is as well observed that there is a sharp drop in the production rate relative to substrate concentration at 0.09. The R squared is seen to be 0.87 which implies that there is a high correlation or association between the production rate and substrate concentration of 87.44%.

Figure 2. Benzoquinone Production Rate versus Substrate Concentration

Discussion

The current study focused on two experiments that included how temperature affects the production of benzoquinone in the presence of catecholase enzyme and how substrate concentration (catechol) affects the production of benzoquinone in the presence of catecholase. The objective of both experiments was achieved through using different techniques where the absorbance readings from the spectrometer were recorded and then analyzed. The analyzed data was able to find out if the results supported the expected outcome or the formulated hypotheses. Based upon the analyzed results, it is evident that the experiments undertaken supported the expected outcome. However, there were some discrepancies observed between the expected outcome and the experimental results and this was associated with the human and experimental errors.

An enzyme has the capacity of undertaking its role optimally when all its specific conditions are in place. In this case, it can’t perform its role accordingly in situations where these specific conditions such as temperature and substrate concentration deviate. Based upon the five experiments (for temperature) undertaken, it was evident that the benzoquinone production catalyzed reactions at 0, 25, 100 and 120 degrees Celsius were relatively slow. On the other hand, at around 50oC the catalyzed reaction for the production of benzoquinone was relatively high of optimum. Enzyme activity increase was predicted by temperature increase and hence expected trend. This was as a result of increased collision that was exhibited between the enzyme and the substrate. In addition, at relatively higher temperatures the prediction was enzyme denaturation (Tijkens, Greiner, Biekman, & Konietzny, 2001). It is evident that between 10 and 30oC, the absorbance was almost constant and as such, at 0oC the absorbance was expected to increase with increase in the production of benzoquinone.

A sharp drop in absorbance is a clear indication of denaturation of the enzyme and this means the enzymatic activity drops sharply. There is a permanent change of the enzyme which has been denatured. This change is as a result of the shape being altered by the heat and hence making it not working appropriately. The heat does not only alter the shape, but as well the level of pH. As was evident in the current experiment, at optimal temperatures, the enzymes work the best. In general terms, the chemical reaction rate is sped up by heat and it is compromised in cases of extreme temperatures.

The results of the five experiments based upon temperature are suggested to support the hypothesis that the reaction rate of catecholase is optimum at temperatures around 37oC which is considered to be normal temperature for human body. In this case, the optimum temperature for the experiment was around 50oC which is closer to 37oC. However, the results showing that the optimum reaction took place at 50oC contradict the formulated hypothesis. This is because it was expected that the enzyme works optimally at 37oC. The absorbance at 50oC is expected to be at around 37oC. This error is suggested to be associated with preparation of the test tubes at very low temperatures (ice condition). Based on these errors, it is suggested that a thermometer should be included when checking temperature accuracy.

The observed results as well as the data are seen to be in agreement with the study undertaken by Dogan, Arslan, and Dogan (2002), who found out that the temperature of the substrate on the enzyme (polyphenol oxidase) led to a slower rate of reaction at temperatures above 40oC as well as below 20oC. However, the current experiment results show some discrepancies (lower rate starts at 50oC) which could be attributed to experimental and human error.

At the point where the temperatures were changed, significant variations were observed in the reactions. Consequently, benzoquinone production was suggested to be optimal at around 50oC. On the other hand, the catalyzed reaction or benzoquinone production was observed to be relatively low at temperatures of 0, 25, 100 and 120oC. The hypothesis is suggested to be supported by these findings where the catecholase functions optimally at temperatures that are around 37oC which is considered to be a normal body temperature.

Despite the fact that the observed data and the results of the experiment undertaken were precise, it could be critical to consider other known enzymes related to the products consumed by humans to confirm and ascertain these results. In this case, there can be consideration of extreme temperatures.

Based upon the substrate concentration results, it can be noted that the benzoquinone production rate is not only affected by the activity of catechol oxidase, but by other factors. In this case, this other factors are temperature, pH, intracellular organization etc. However, substrate concentration was considered in the current experiment. Based upon cellular organization, it can be pointed out that benzoquinone production relied upon the level of the available enzyme in the given solution. For the solution the solution that never had the enzyme for example, benzoquinone production was seen to be relatively slow. This was considered to be slower than that samples that contained the enzyme. It was noted that the sample that had the highest enzyme amount had a pronounced reaction. The graph shape in this case can be explained at a molecular angle as: There is was production of zero benzoquinone in the course of oxidization of the catechol. Later, melanin was formed as a result. The color of the solution became deeper (from pink to brown) as the size of the polymer increased. These molecules will precipitate out of the given solution because of their less solubility in water. The precipitation process is spontaneous initially as evident in the results section. Thus, the process decreases at the last stages of the reaction. This observation is in agreement with the study undertaken by Campbell and Reece (2002) where the enzymatic activity increased with increase in substrate concentration up to a point where it leveled off ( termed as saturation point). The graph in the result section clearly depicts this expected outcome where increase in substrate concentration increased with increase in the activity of the enzyme.

AS noted in the formulated hypothesis; the reaction rate increases with increase in the substrate concentration. The enzymatic activity was seen to increase at every substrate concentration level. However, this increase became horizontal and this is where the substrate has occupied all the available enzymes and it is termed as plateau with maximum rate of reaction. There is increase of benzoquinone production with increase in substrate concentration over a given period of time. This can be explained as the reaction takes place with time.

Certain errors could be noted despite the fact that the results of the experiment were a reflection of what was expected. Some data points for instance could not be used in generating the graph because they were considered to be outliers. It is as well pertinent to note that the rate of reaction at 0.2mL concentration point was higher when compared to that of 1mL and 0.5mL. This is considered to be as a result of experimental error because substrate concentration should increase with increase in the rate of reaction. This observed error is however seen not to be consistent because other data points followed the trend as expected. It can be suggested that the error could be associated with human errors such as incorrect measurements of solutions and reagents. The experiment is suggested to be reliable because the results were according to the expectation.

Substrate concentration was another variable studied in the current experiment. The substrate concentration was increased in different levels (0.2, 0.5, 1, 2.5 & 3 mL) to evaluate enzyme activity. Based upon this, a hypothesis was formulated as: the reaction rate increased with increase in the substrate concentration. The increase in the concentration of the substrate was maximal at the time where the substrate has occupied all the enzymes. The formulated hypothesis reflects the predictions of the experiment.

The reaction observed in the current experiment entailed catechol conversion to benzoquinone and thus it was facilitated by polyphenol oxidase enzyme (Creveling, 2000). The experiment results were used in undertaking different analyses accordingly. The catechol color changed in the course of the experiment.

Enzymes have an optimal operating temperature range that allows them to function at their highest level. The temperature of the human body () is the perfect range for these enzymes to operate at maximum efficiency. At higher temperatures the enzymes effectiveness decreases and they begin to break down. The current experiment was designed to measure the factors that cause an enzymes catalyzed reaction. The enzyme catecholase was used for this purpose, with the desire to create the end product benzoquinone. The catecholase was used as a catalyst to create a reaction in the substrate turning it to benzoquinone which is reddish brown in color. This change enabled the determination when the colorless substrate had changed into Benzoquinone.

Methods

This section explains how the two experiments were undertaken and in this case they were based on the effect of temperature and substrate concentration on the production of benzoquinone. Thus, the main objective was to determine the impact of substrate concentration (catechol) and temperature on catecholase (or benzoquinone production). In addition, the two experiments aimed at predicting the expected outcome where it was expected that the catecholase works optimally at 37oC. This was evaluated based on the temperature range of 0oC to 100oC. On the other hand, to determine how substrate concentration affected benzoquinone production, different concentrations of samples of the substrate were considered. This was evaluated against absorbance as evident in the subsequent sections. The method section was therefore divided into two subsections and they included temperature and substrate concentration.

Temperature

Benzoquinone production rate measurement in the presence of the catecholase enzyme was achieved by using a spectrophotometer. In this case, the solution absorbance was read in the spectrophotometer where the wavelength was set at 540nm. A higher concentration of benzoquinone resulted to a darker solution and hence a higher absorbance. Thus, the measurement of reaction rate was by absorbance increase with time. Solanum tuberosm (mashed potato) was used in the preparation of catecholase where 20 grams of it was put in 500 ml of water. A grease pencil was used to label 3 tubes as 1, 2, and 3. Labeling of the 3 pipettes was undertaken as catechol, potato and water and this was done to avoid cross contamination during the process of measuring. The first step was making of the blank which consisted of 9ml of distilled water and 3ml of potato extract (they were put in tube 1 after measurement) (Table 1). Secondly, the measured catechol (4ml) was put in tube 2(Table1). The third step entailed measuring distilled water (5ml at 25oC) and extract of the potato (3 ml) in tube 3(Table 1). The spectrometer was zeroed out using part of the solution of tube 1 where it was placed in a cuvette. Later, the cuvette was emptied and then rinsed using distilled water before drying it upside down on a towel. Read More