ne Catalyzed by Varying Concentrations of Catechol Oxidase as a Function of TimeABSTRACT
The rate of the reaction of catechol to benzoquinone was tested using varying concentrations of catechol oxidase. The experiment setup consisted of four different test tubes containing a varying concentration of the enzyme catechol oxidase. Each sample was placed in a spectrophotometer set at 500 nm. Absorbance readings were taken every 15 seconds. The analysis of variance indicated that the amount of enzyme present was highly significant. It was determined that the rate of reaction of catechol to benzoquinone increased as the amount of enzyme increased.
In many chemical reactions, there is a transfer of one or more electrons from one reactant to another. These electron transfers are called oxidation- reduction reactions, or redox reactions for short. In a redox reaction, the loss of electrons from one substance is called oxidation, and addition of electrons to another substance is known as reduction. Because an electron transfer requires both a donor and an acceptor, oxidation and reduction always go together (Biology 1999).
Compounds that have a hydroxyl group directly attached to a benzene ring are called phenols. This is the specific name for a substance known as hydroquinone, which can be oxidized to produce a compound known as benzoquinone; therefore, benzoquinone can be reduced to form hydroquinone (Enzyme Chemistry 1955). Our experiment was the oxidation of a hydroquinone, commonly known as catechol, to the compound benzoquinone.
In order to increase the rate of reaction, an enzyme was used. The enzyme was catechol oxidase (potato extract). Enzymes are catalytic proteins that speed up reaction without being consumed by the reaction. Every chemical reaction has an energy barrier known as activation energy. This is the amount of energy for starting a reaction. An enzyme actually lowers these energy barriers and speeds up the reaction (Enzyme Chemistry 1955).
The purpose of this study was to determine the absorbance or the rate of reaction of catechol to benzoquinone catalyzed by varying concentrations of catechol oxidase (enzyme). It was hypothesized that the amount of catechol oxidase in the reaction would increase the rate of reaction. If this were true, then the solutions with the most enzyme in them would have the highest absorbance rate.
MATERIALS AND METHODS
Four large test tubes were each filled with 1 ml. of distilled water + 5 ml. of catechol. One large test tube was prepared with 2.5 ml. of potato extract, which was our enzyme, and labeled Extract. Three more large test tubes were filled with 9 mls. of distilled water. One ml. was taken from the tube labeled Extract and placed in the first tube with 9 mls. of distilled water. This is the one that was labeled 1/10. Next 1 ml. from the 1/10 dilution tube was taken and placed into the second tube with 9 mls. of distilled water, and labeled 1/100. Finally 1 ml. from the 1/100 dilution tube was taken and placed into the third tube with 9 mls. of distilled water, and was labeled 1/1000. These were the four dilutions that would be mixed with the enzyme and placed in the spectrophotometer, which would measure the rate of reaction (absorbance).
The experiment was to measure the rate of reaction between the extract and the four extract + water dilutions. One ml. was taken from each and placed in the spectrophotometer. Then the rate of reaction or absorbance was recorded off the spectrophotometer for two and one half minutes, taking a reading every 15 seconds. The experiment is the rate of the reaction of catechol to benzoquinone catalyzed by varying concentrations of catechol oxidase (potato extract) as a function of time.
Our independent variable was the amount of potato extract in each of the four solutions. The dependent variable was the rate of reaction of the four solutions (catechol) into benzoquinone. The data was graphed using Microsoft Excel on the computer. The graph was the absorbance of the reaction vs. the time of the reaction (figure 1). There were four sets of graph data and four lines.
My partner and I performed the experiment of the rate of the reaction of catechol to benzoquinone catalyzed by varying concentrations of catechol oxidase as a function of time. Our graph (figure 1) is a line chart with time (s) as our x-axis and absorbency (ODU) as our y-axis.To measure the rate of the reaction, which is absorbance/time, we used a spectrophotometer measured in 500 nm of light. Absorbance, which was the measure of the amount of benzoquinone in the solution, increased on all of the solutions except 1/100, which showed no change in absorbance. As you can see from our graph (figure 1), from greatest to least, the range of absorbance from 0 to 150 seconds was: Full=.599-.766; 1/10=.066-.143; 1/100=.011-.019; 1/1000=.003-.005.
The maximum absorbance was .766 by the solution with the highest enzyme concentration. Our minimum absorbency was .003 by the solution with the lowest enzyme concentration. We had a positive slope because the absorbency was always increasing between every fifteen-second calculation. All of our concentrations increased the most rapidly from zero to thirty seconds. From there, they gradually increased until the end. As the reaction was performed, the clear solution (catechol) became darker and yellow (benzoquinone).
The results from our experiment supported our alternative hypothesis which was the absorbency was affected by the amount of catechol oxidase that was involved in the reaction. We rejected our null hypothesis which stated that the amount of catechol oxidase did not affect the reaction. Our experiment was the oxidation of a hydroquinone (catechol) to produce the compound benzoquinone. This oxidation was actually the removal of one pair of electrons and two protons from the hydroquinone. The reaction is a reversible process; the benzoquinone can be easily reduced by mild reducing agents to hydroquinone in enzyme-catalyzed reactions.
Enzymes are catalytic proteins that increase the rates of chemical reactions by reducing the activation energy, without being consumed by the reaction (Biology, 1999, 97-102). Knowing this in our experiment, we used various concentrations of the enzyme catechol oxidase (potato extract) and observed a gradual increase in the formation of benzoquinone. Without enzymes, the chemical pathways of metabolism get congested. The enzyme holds a substrate and a coenzyme together so the oxidation reaction can take place. The coenzymes most commonly used by the enzymes that catalyze oxidation reactions are aclotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) (Outlines of Enzyme Chemistry, 1958).
With this knowledge about enzymes, we figured that the concentration with the most potato extract would have the highest absorbency. Our experiment went well, and we did not have any major problems. Our results were accurate with what we expected to get, based on our knowledge of enzymes. The solution with the highest concentration of enzyme had the highest absorbency, and then the solution with a little less concentration of enzyme was lower, and so on.
The Rate of the Reaction of Catechol to Benzoquinone Catalyzed by Varying Concentrations of Catechol Oxidase as a Function of Time
Biology Laboratory for Science Majors (BIOL. 1208)