How to Measure Enzyme Activity
What is enzyme activity? This article will discuss how to measure enzyme activity, the units of enzyme activity, and the effects of pH and temperature on enzyme activity. We will also discuss the importance of enzyme activity in biological research. Let’s begin! How to Measure Enzyme Activity
Measurement of enzyme activity
The measurement of enzyme activity requires the presence of other proteins, such as a substrate. The measured initial velocity is normalized to the total protein concentration, which is expressed as weight per volume. The molar units of the initial rate are divided by the weight of the complete protein to obtain the specific enzyme activity. The specific enzyme activity is then expressed as the number of moles of product converted per unit time per unit protein weight.
The measurement of enzyme activity has many applications, from discovering and analyzing damaged tissue to biochemistry and neurochemistry. Fluorescence techniques can be used to generate a quantitative histological image of enzyme activity, and near-IR fluorescent probes have been adapted for in-vivo work. Magnetic-resonance-based methods have also been used to investigate enzyme activity. And finally, mass spectrometry (MALDI) can be used to obtain images of diseased tissues and understand pathological processes.
The specific enzyme activity is quoted in units per ml or nmol/ml. The units per ml measurement are equivalent to nmol/min per ml. This value is useful in comparing enzyme purity, as values obtained for different batches of pure enzyme should be comparable within the margin of error. For example, two enzyme solutions may contain the same number of units but have completely different specific activity values.
Units of enzyme activity
Units of enzyme activity are defined as the amount of substrate an enzyme catalyzes per unit of time. The SI unit for enzymatic activity is the katal, equal to one mol S-1. In practice, enzyme activity is usually expressed in enzyme units (U), which are one million times smaller. Enzyme activity is also measured for other standardized substrates, such as milk proteins or gelatin.
The Unit is derived from the SI unit katal and measures the enzymatic activity of enzymes and other catalysts. It should not be confused with reaction rate, measured in moles per liter of the substrate. Instead, katal is a measure of catalytic activity. The katal measures enzyme activity but does not describe the rate of reaction. However, katal is useful for measuring enzyme activity.
The Nomenclature Committee on Enzymes introduced the unit E in 1961. This unit has derivatives: specific activity, molecular activity, enzyme catalytic center activity, concentration in solution, and catal. The information provided is essential for the proper expression of enzyme activity. The following paragraphs describe the steps involved in evaluating the corresponding units. Once you have identified the important parameters for the assay, you can further analyze the results.
The units of enzyme activity are expressed as katal/kg, U, or mmol per minute. For convenience, activity is usually expressed in nmol per ml, rather than units per ml. The latter is more convenient, but the larger volume will have double the amount of product. Therefore, it is important to choose the corresponding unit when evaluating the results. The nmol/ml units of enzyme activity will serve as a guideline when allocating internal activities costs.
Effect of temperature
The effect of temperature on enzyme activity can be studied in many ways. For example, the enzyme can be a large protein macromolecule. The higher the temperature, the more probable the substrate will fit into the active site. The higher the temperature, the faster the reaction will proceed. This effect is also useful in biochemical engineering. However, the enzyme must be activated at a high temperature for operational reasons in many cases. In such cases, temperature dependence can be useful in the wrong situations.
The Equilibrium Model is a good description of the temperature dependence of enzyme activity. The Equilibrium Model is based on two important parameters that describe the thermal behavior of enzymes. The midpoint temperature is related to the organism’s growth temperature, and the enthalpy of equilibrium is its ability to function over different temperature ranges. This model also describes the difference between active and inactive forms of an enzyme at its active site.
The more increased the temperature, the quicker the enzyme reacts. However, the higher the temperature, the higher the chances of the enzymes colliding with each other. Too high a temperature can denature the enzyme, making it incapable of binding to substrates. For this reason, the temperature is an important factor in enzyme activity. This is one of the numerous essential factors for understanding how to use enzymes. And a high temperature can be even worse for your enzyme.
Effect of pH
An enzyme’s activity is influenced by its pH and substrate structure. At low pH, the substrate cannot bind to the active site, and the enzyme is unable to generate a product. This result can be reversible as the enzyme can return to its original pH after it changes. The pH of an enzyme is the determining factor for its activity. Hence, maintaining a constant pH is important to ensure the desired outcome of the reaction.
The pH of a solution affects the rate of enzyme-catalyzed reactions. Most enzymes exhibit a maximum speed at a certain pH. This peak pH is called the pH optimum. This profile is a graph of the rate against pH. It has no absolute significance and varies according to the enzyme studied and the measurement conditions. This graph is useful for studying the speed of an enzyme at different pH levels. A pH optimum is often defined as the pH level at which the enzyme has a maximum activity in practical applications.
The pH level of a solution depends on the species of Trichoderma used for fermentation. In this study, chitosanase enzymes were produced by Trichoderma spp. in both acid and base medium. The effect of pH on enzyme activity was statistically significant for all strains. However, for T. viride, the pH effect was stronger and quadratic than for the other strains.
Effect of protein concentration on enzyme activity
The concentration of an enzyme determines its activity. The higher the concentration, the faster the reaction. This is because the concentration of the enzyme is proportional to the substrate concentration. In the graph below, the concentration of enzymes is plotted against the reaction rate. As the enzyme concentration increases, the number of active sites increases. The enzyme will only work at its maximum rate if all of its substrate molecules are bound to it.
In addition, the enzyme’s conformation and primary structure may be affected by extreme pH environments. The optimum pH for an enzyme is different from its intracellular surroundings. In these cases, the pH may exert a controlling influence on the enzyme’s activity. Hence, it is important to know the pH conditions of enzymes before using them. To help us understand the mechanism behind these varying conditions, we need to look at how enzymes function.
Enzyme assays are generally linear up to an OD of 2.5. The dilution factor of 0.02 produces a strong signal and lies in the middle of the linear range. However, calculations using a dilution factor of 0.04 to 0.12 will underestimate the enzyme’s real activity. To avoid this problem, enzymes must be prepared at known concentrations. This ensures reproducibility.
Calculation of enzyme activity
The most common method for calculating enzyme activity is to use a spectrophotometric assay. The enzyme activity of a sample is measured by measuring the amount of signal (in units per mL), which varies with the increasing concentration of the substrate. This process requires a standard curve calculated by measuring the assay signal with a standard solution. For example, if the enzyme activity of a sample is equal to the activity of a gram of protein, the resulting value will be 50% higher than that of the same volume.
This procedure is more accurate than other methods because the activity of enzymes is more specific. In addition to considering the spectrophotometric data, it is also important to consider the assay controls. For quantitative work, a high signal-to-background ratio is essential. To achieve a high signal-to-background ratio, varying assay parameters are often necessary. In addition, varying enzyme concentrations may not always be sufficient.
High-throughput microplate techniques allow researchers to examine the enzyme activity of a large number of samples simultaneously. These methods allow the measurement of enzyme activity and other environmental parameters for a wide variety of samples. Additionally, the method enables the comparison of multiple samples at the same time, reducing the overall reagent volumes per sample. A microplate method has been used to analyze enzyme activity for over 30 years and is the most widely used method for measuring enzyme activity.