PPO is an enzyme found widely across the biological world, occurring in plants, fungi, and bacteria. It is best known as the agent responsible for the rapid browning that occurs when certain fruits and vegetables are cut or bruised. PPO acts as a catalyst, accelerating the oxidation of naturally occurring organic compounds called phenolic compounds. The enzyme itself is a protein that contains copper atoms at its active site, which are necessary for its function as an oxidase.
The Enzymatic Reaction Causing Browning
Enzymatic browning requires three components: the PPO enzyme, a phenolic compound substrate, and molecular oxygen. In the intact plant cell, the enzyme and its phenolic substrates are kept physically separated in different cellular compartments, such as the chloroplasts and vacuoles. This separation prevents premature reaction. When the plant tissue is damaged, this cellular separation is destroyed, allowing the PPO and the phenolic compounds to mix freely.
The reaction begins when PPO uses oxygen to oxidize phenolic compounds, which contain a hydroxyl group attached to an aromatic ring structure. PPO specifically acts on \(o\)-diphenols, which are phenolic molecules with two hydroxyl groups positioned next to each other. The enzyme converts these colorless phenolic substrates into a group of highly reactive molecules known as \(o\)-quinones. This conversion is a rapid process that consumes the available oxygen in the immediate vicinity of the wound.
These newly formed \(o\)-quinones are unstable and react immediately with other molecules present in the damaged cell. They spontaneously undergo a non-enzymatic reaction called polymerization, where individual quinone molecules link together to form long, complex chains. This process results in the formation of large, pigmented compounds known as melanins. Melanins are the dark-colored polymers responsible for the brown, black, or sometimes red pigments visible on the exposed tissue surface.
The speed of the browning reaction depends on the concentration of PPO and phenolic compounds in the tissue, as well as the local temperature and \(\text{pH}\). Foods like potatoes and apples contain high levels of phenolic compounds, making them highly susceptible to browning. The initial formation of quinones is a direct result of the enzyme’s activity, but the final visible color change results from the subsequent chemical polymerization of the quinones.
PPO’s Purpose in Plant Defense
While PPO is often viewed negatively due to its role in food spoilage, its function in a living plant is entirely protective. The enzyme is a fundamental part of the plant’s defense system against external threats and physical damage. The browning reaction acts as an immediate biological response mechanism to wounding, such as from a cutting tool or an insect pest.
When a plant is wounded, the mixing of PPO and phenolic compounds triggers the rapid production and subsequent polymerization of quinones. The resulting melanin polymers create a physical barrier, effectively acting as a scab to seal the injured site. This seal helps the plant prevent water loss and blocks the entry of harmful microorganisms and pathogens into the exposed tissue.
The highly reactive quinones produced during the PPO-catalyzed reaction are also toxic to many organisms. These quinones can react with and bind to proteins and amino acids in the digestive systems of insects and herbivores. This chemical modification reduces the nutritional quality of the plant tissue, making it less palatable or digestible to the attacking organism. The activation of PPO is therefore an induced defense, a chemical weapon deployed only when the plant is under attack.
Methods for Inhibiting PPO Activity
Controlling enzymatic browning requires interrupting one of the three components necessary for the reaction: the enzyme, the substrate, or the oxygen. Practical methods for inhibition focus on altering the environment to make it unsuitable for PPO activity or by chemically interfering with the reaction intermediates.
One of the most effective methods is to exclude oxygen, since it is a required co-substrate for the PPO enzyme. Submerging cut produce in water, oil, or syrup creates a physical barrier that prevents atmospheric oxygen from reaching the exposed tissue surfaces. Commercial practices often use modified atmosphere packaging or vacuum sealing to achieve a similar, more complete exclusion of oxygen from the product.
A successful strategy involves reducing the \(\text{pH}\) of the environment through the application of mild acids. Polyphenol oxidase has an optimal activity range between a \(\text{pH}\) of \(5\) and \(7\), which is common in many fruits and vegetables. Applying agents like lemon juice or vinegar lowers the \(\text{pH}\) to an acidic level, typically below \(4\). This shift in \(\text{pH}\) causes the enzyme’s structure to change, effectively slowing its catalytic function.
Heat treatment is a way to permanently stop PPO activity through a process called denaturation. Enzymes are proteins, and exposing them to high temperatures causes their three-dimensional structure to unravel, rendering them biologically inactive. Brief boiling, known as blanching, is commonly used in commercial food processing to inactivate PPO before freezing or canning vegetables.
While heat permanently deactivates the enzyme, simply lowering the temperature, such as by refrigeration or freezing, only slows the reaction rate. Enzymes operate more slowly in colder conditions, meaning the browning process will take longer to occur, but it will not be stopped entirely. For long-term storage, this is often paired with other inhibition methods to achieve better results.
Finally, certain chemical compounds can interfere with the reaction, such as the use of reducing agents like ascorbic acid, also known as Vitamin C. Ascorbic acid does not stop the PPO enzyme itself but instead reacts with the \(o\)-quinones as soon as they are formed, converting them back into their original, colorless phenolic state. This essentially neutralizes the reactive intermediates before they can polymerize into the brown melanin pigments.