Aim
The experiment aims to study the effect of substrate concentration on the activity of polyphenol oxidase (PPO) from banana extracts using spectrophotometric techniques.
Introduction
An enzyme refers to a molecule that is protein in nature and works as a biological catalyst. The enzyme possesses three major characteristics. The first characteristic is that they act to increase the rate at which a given reaction takes place. This enables most cellular reactions to take place at up to million times faster compared to their rate in the absence of an enzyme. Secondly, most enzymes act specifically on a single reactant known as a substrate to produce the products. The last characteristic is the fact that the activity of the enzymes is regulated from low to high activity and vice versa (Gregory, 2013).
The specificity of the enzymes varies with some enzymes having an absolute specificity for acting on only a single substrate while others may act on various substrates that have similar side chains, functional groups, or locations on a chain. Those enzymes that are least specific are those enzymes that act on a given chemical bond irrespective of the other features of the structure (Gregory, 2013).
The substrate is able to interact with the active site using the hydrogen bonding, opposite charges, hydrophobic non-polar interaction, as well as the coordinate covalent that bond to the metal ion activator. The way through which the enzyme and substrate interact can be described using two theories. These theories are the lock and key theory and the induced fit theory (Gregory, 2013).
The lock and key theory states that the enzyme acts as the lock and can only be acted upon by a specific key which is the substrate. Only the correct key or substrate will be able to fit into the hole of the lock or enzyme to open the lock or result to a reaction. Larger, keys, smaller keys, or even the keys that have teeth that are incorrectly positioned are not able to fit into the lock (enzyme). It is only the keys that have the correct shape that are able to open a specific lock. Only the key that are correctly shaped opens a particular lock. The substrate must, therefore, fit into the shape of the enzyme for the reaction to be catalyzed (Vasudevan, 2007).
In the induced fit theory, it is assumed that the substrate works to determine the final shape to be assumed by the enzyme. This also suggests that the shape possesses by the enzyme at the active site is flexible. This may explain the reason why some compounds are able to bind the enzyme but are not able to react since the enzyme structure is distorted. Other molecules may be too small and thus unable to induce the appropriate enzyme alignment and, therefore, no reaction. It is only the proper substrate that has the capability to induce the appropriate alignment of the enzyme at the active site. The induced fit theory provides a better explanation to the interaction between the enzyme and substrate since some of the experimental evidence cannot be explained adequately through the rigid lock and key theory (Vasudevan, 2007).
Polyphenol oxidase or PPO refers to a tetramer which is made up of four copper atoms per molecule and two distinctive binding sites for the oxygen molecules and aromatic compounds. The enzyme works to catalyze the hydroxylation of those phenol molecules that have a benzene ring with a single hydroxyl substituent forming diphenols (Worthington Biochemical Corporation, 2011). A diphenol molecule is the one that two hydroxyl substituent. The polyphenol oxidase may further catalyze orthodiphenol oxidation to form orthoquinones. The orthoquinones formed undergo rapid polymerization to form polyphenols that are brown, black, or red in color. The polyphenols are responsible for the browning of the fruits. Amino acids such as the tyrosine have a single phenolic ring. The ring may undergo oxidization through the action of PPOs forming the orthoquinone. The action of PPOs on tyrosine leads to the naming of PPOs as a tyrosinase (Mayer, 2006).
The PPOs has a molecular weight of 128,000 (kD) and works at an optimum pH of 6-7. The extinction coefficient immediately after the enzyme is purified is 24.9, and its activity is mainly inhibited by those compounds that can make a complex with copper, as well as benzoic acid and cyanide. The specificity of PPOs is mainly on most of the catechols that are parasubstituted.
In bananas, the natural substrate of PPO is the dopamine. The dopamine in bananas is oxidized by the PPOs to form dopamine quinine, which is then oxidized spontaneously to form a red product known as 2,3-dihydroindole-5,5 quinone. The reaction process from dopamine to red quinone is as shown the equation below. The red quinine that is formed enables PPO to be assayed conveniently using a spectrophotometer.
Methods
The laboratory protocol was followed.
Results
The absorbance readings for the two replicates were recorded and their average determined as shown in Table 1 below.
Using the data recorded, a graph of average absorbance against time was plotted to show the relationship between the two variables as shown in Figure 1 below. The absorbance increased as time progressed. The absorbance readings at any given time were highest in the reaction where the substrate (banana extract) has the highest concentration and lowest where the concentration of the substrate was lowest.
Figure 1: Relationship between absorbance and time at varying banana extract amount
Using the extinction coefficient of red quinone, (2515 mol-1Lcm-1 at 470 nm), the rate of product production in mM/min/ml may be calculated. According to the Beer's Law, the absorbance of the substrate is directly proportional to the amount of the substrate in a sample. The absorbance also depends on the thickness of the sample according to the Lambert's Law. Combining the two laws gives the Beer-Lambert law which can be illustrated using an equation as follows.
A=εcl
The ε in the equation is the extinction coefficient; c is the concentration of substrate usually in mol/L while the l is the thickness of the sample which is given in cm. Using the above equation, the concentration of red quinone at 0 seconds and 30 seconds were calculated for each volume of banana extract that was added and the rate of red quinone formation calculated from the two values. The calculations were done as follows.
For the reactions where 10μL was added, the concentration of red quinone at 0 seconds was
A=εcl
c=Aεl
=0.02152515×1
=8.549 mM/ml
The concentration of red quinone after 30 seconds was
A=εcl
c=Aεl
=0.082515×1
=31.81 mM/ml
The amount of red quinone formed within the 30 seconds was
31.81-8.55
=23.26 mM/ml
The rate of red quinone production =23.26 mM/mL0.5 minutes
=46.52mM/min/mL
For the reactions where 20μL was added, the concentration of red quinone at 0 seconds was
A=εcl
c=Aεl
=0.0245×1062515×1
=9.742 mM/ml
The concentration of red quinone after 30 seconds was
A=εcl
c=Aεl
=0.09252515×1
=36.78 mM/ml
The amount of red quinone formed within the 30 seconds was
36.78-9.74
=27.04 mM/ml
The Rate of red quinone Production =27.04 mM/mL0.5 minutes
=54.08 mM/min/mL
For the reactions where 40μL was added, the concentration of red quinone at 0 seconds was
A=εcl
c=Aεl
=0.0205×1062515×1
=8.151 mM/ml
The concentration of red quinone after 30 seconds was
A=εcl
c=Aεl
=0.1362515×1
=54.076 mM/ml
The amount of red quinone formed within the 30 seconds was
54.076-8.151
=45.925 mM/ml
The Rate of red quinone Production =45.925 mM/mL0.5 minutes
=91.85 mM/min/mL
For the reactions where 60μL was added, the concentration of red quinone at 0 seconds was
A=εcl
c=Aεl
=0.027×1062515×1
=10.736 mM/ml
The concentration of red quinone after 30 seconds was
A=εcl
c=Aεl
=0.1782515×1
=70.775 mM/ml
The amount of red quinone formed within the 30 seconds was
70.775-10.736
= 60.039 mM/ml
The Rate of red quinone Production =60.039 mM/mL0.5 minutes
=120.078 mM/min/mL
For the reactions where 80μL was added, the concentration of red quinone at 0 seconds was
A=εcl
c=Aεl
=0.0465×1062515×1
=18.49 mM/ml
The concentration of red quinone after 30 seconds was
A=εcl
c=Aεl
=0.24652515×1
=98.012 mM/ml
The amount of red quinone formed within the 30 seconds was
98.012-18.49
=79.522 mM/ml
The Rate of red quinone Production =79.522 mM/mL0.5 minutes
=159.044 mM/min/mL
The results obtained can be summarized as shown in Table 2 below. The results indicate that the initial rate of reaction was lowest when the quantity of banana extracts that were added was low and highest when the amount of banana extracts was high.
The information on the amount of banana extracts that were added and the initial rate of reaction at every concentration was used to plot a graph of rate of reaction in change, in absorbance measured at 470 nm against the volume of banana extract that was added. The graph shows that the rate of reaction increased as the concentration of the banana extracts increased.
Figure 2: Relationship between the rate of reaction and the amount of banana extract added
Discussion
The activity of polyphenol oxidase has been studies widely especially in banana fruits due to its role in the enzymatic browning effect witnessed in the banana plant. The rapid loss of color in the leaves, roots and stem tissues after the banana plants are injured and the strong tissue pigmentation after extraction as indications that PPO together with the phenol compounds are ubiquitous not only in the banana tissues but also in vegetative tissue of the banana. Once the banana is injured, the injured tissues are exposed to the air leading to a rapid tissue browning. The pigmentation is as a result of the oxidation of the phenol compounds by the PPO forming the quinones. The formed quinones then undergo polymerization into polyphenols. The polyphenols lead to the binding of the proteins, DNA and RNA altering the protein conformational structure. This inactivates the enzymes, and, as a result, there is a drastic alteration in the profile of the extracts (Wuyts, De Waele, & Swennen, 2006).
The activity of an enzyme is determined by factors such as the enzyme and substrate concentration, pH and temperature. When these factors are lower or higher than the optimum level, the activity of an enzyme reduces. The activity of the enzyme, therefore, requires an optimum level of these factors for the activity to be optimum. In this experiment, the amount of the substrate (dopamine) in the banana extract was the independent variable. As the amount of the substrate increased, the rate of the enzyme increased.
For the enzyme to be able to catalyze a chemical reaction, the substrate first binds to the enzyme. The binding is achieved through the random collisions that take place between the substrate and the enzyme as the particles move randomly in the solution. The frequency of collision may be affected by the amount of the substrate compared to that of the enzyme in a reaction solution. The higher concentration of the substrate in a given reaction, the higher the chances for a collision between the substrate and the enzyme occurring. The collisions also need to take place at the right orientation to ensure that the substrate collide with the enzyme on the side where the active site is located (Finkler, 2005).
There is, however, an indefinite level at which an increase in substrate concentration may lead to an increase in enzyme activity. Once the enzyme has collided with the substrate and the two bind together, it takes some time for the substrate to be processed to the final product. There is thus a given amount of substrate that can be catalyzed by the enzyme available at a given time. As the concentration of the substrate increases, there are an increased number of enzyme molecules that are engaged in enzyme-substrate complexes at any specific time leading to saturation of the enzyme at a given substrate concentration. This means that there will be no further increase in the activity of the enzyme as the substrate concentration is increased. This explains the reason why the enzyme activity reaches a plateau at higher substrate concentration. In a given reaction, there was a rapid increase in absorbance, and after some time the absorbance reaches a plateau. The plateau is reached at a point where the substrate has been exhausted, and no more substrate is being formed (Bassiri, 2013).
In conclusion, the experiment aimed to study the effect of substrate concentration on the activity of polyphenol oxidase (PPO) from banana extracts using spectrophotometric techniques. Through the spectrophotometric techniques applied, the activity of PPO in oxidizing dopamine was successfully determined and the effect of substrate concentration determined. The study showed that, at concentrations of the substrate, the activity of the enzyme is directly proportional to the amount of the substrate added.
Reference List
Bassiri, E. (2013). Enzyme Kinetics Theory. Retrieved March 27, 2014, from http://www.sas.upenn.edu/LabManuals/biol123/Table_of_Contents_files/8d-EnzymeKinetics-Theory.pdf
Finkler, M. S. (2005). Lab #4: Enzymes. Retrieved March 27, 2014, from http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab4.pdf
Gregory, M. J. (2013). Energy and Enzymes. Retrieved March 26, 2014, from http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/bio%20101/bio%20101%20lectures/energy/energy.htm
Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry, 67(21), 2318-2331.
Vasudevan, D. (2007). Textbook of Biochemistry for Dental Students. New Delhi: Jaypee Brothers Publishers.
Worthington Biochemical Corporation. (2011). Polyphenol Oxidase. Retrieved March 26, 2014, from http://www.worthington-biochem.com/TY/default.html
Wuyts, N., De Waele, D., & Swennen, R. (2006). Extraction and partial characterization of polyphenol oxidase rom banana (Musa acuminataGrande naine) roots. Plant Physiology and Biochemistry, 44(5), 308-314.
