Enzymes are a special kind of protein found in cells of living organisms. They’re made up of long chains of amino acids held together by peptide bonds. No two types of enzymes have the same amino acid structure, and each enzyme has its own unique shape. Automation of enzyme assays is becoming increasingly important and instrumentation is being developed to satisfy this need.
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Usually,Discrete the assay is carried out by determining the enzyme activity with, and without activation by an added coenzyme. The activity can be monitored by measuring changes in concentration of substrates or products during the reaction. Enzyme activity is the rate of enzyme reaction — generally expressed as units of substrate converted (or product formed) per time unit. Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes.
Factors affecting enzyme assay analysis
Measuring enzyme activity is a precise job and can be influenced by many variables. Results accuracy is highly dependent on temperature stability. Just a one-degree temperature change can lead to a 4–8% variation in enzyme activity. For consistent and reproducible results, an enzyme assay should be carried out in well-defined conditions that can be duplicated in other laboratories. Variables such as pH and buffer type, ionic strength, and temperature must be strictly controlled. pH is a critical parameter in method development and routine enzyme assay measurement.
pH affects the enzyme activity, charge, and shape of the substrate so that the substrate cannot bind to the active site or cannot be catalyzed to form a product. All enzymes have an ideal pH value, which is called optimal pH. Under the optimum pH conditions, each enzyme showed the maximum activity. Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Fixing the other variable will allow correlating the change in measuring parameter and absorbance directly to the enzyme assay or enzyme activity. Reliable enzyme assay development is critical and the automated enzyme analyzers simplify the overall method development and results reliability.
Enzyme assays — what are the method choices?
Most enzyme assays are based on spectroscopic techniques, with the two dominant types being absorption and fluorescence. The spectrophotometric assay is a classic enzyme test, which remains as the most widely used assay for the lowest cost. All the steps involved are manual; controlling several variables manually leads to inconsistent results and makes the overall method development tedious and unreliable. This method is suitable when analyzing a few samples or enzyme-type routine operations.
Enzyme assays based on photometry, fluorometry, 96-, 384-, or even 1536-well format microplate offer a high-throughput alternative to the traditional spectrophotometers. The microplate format is convenient for high-throughput analysis using a 200 μL assay volume and are commonly used in life science applications. However, the microplate method suffers from temperature stabilization, absorption correction, and edge effect. The absorbance is measured vertically on microplates through the well, so several factors affect the liquid pathlength, and thus the absorbance. Therefore, in photometric microplate measurements, pathlength correction is required for calculating the enzyme assay. The primary cause for the “edge effect” phenomenon is evaporation and is commonly associated with 96-well microplates. Edge effect is an issue attributed to the increased evaporation rate of circumferential wells compared to centrally located wells. Often a great deal of work goes into assay development. They have limited incubation temperature, temperature stability, and precision, which limits the application range.
For some enzyme assays, it is necessary to quench or stop the reaction at a specific time to prevent further production of the product. For example, samples may be taken at 5-minute intervals for a predetermined time with the product being measured by high-performance liquid chromatography (HPLC). Each chromatographic analysis may take 30 minutes to complete.
Fully automated enzyme assay analysis
In comparison to the spectrophotometer or direct read microplate systems, the Gallery Plus discrete analyzers offer a wide incubation temperature range from 25° C to 60° C expanding the application possibilities. All the substrate additions and measurements are done in disposable low-volume cuvette, allowing the system to perform real-time kinetic measurement.
Superior temperature control and lack of edge effects ensure confidence in results. Thanks to dedicated software, enzyme workflows are incredibly simple with practically no change over time from one method to another. With flexible method parameters for each enzyme type — measuring wavelength, blank measurement, buffer addition, reagents additions, substrate addition, enzyme specific incubation temperature, enzyme specific incubation time, and data collection duration-the enzyme assay method development and transfer are effortless and reliable from R&D to QA/QC labs. Optional electrochemistry unit measures Optimal pH of the samples and helps to maintain consistent experimental condition.