Comparison of Enzyme Activity Assay Methods

Enzyme activity assay is an analytical technique that uses the properties of enzymes to specifically and efficiently catalyze chemical reactions, and detects the content and activity of certain enzymes in biological samples such as body fluids by measuring the rate of enzymatic reactions. 1. Classification by reaction time:rnBefore the 1950s, the fixed-time method was mostly used. This method is based on the average speed of the enzyme-catalyzed reaction to calculate the enzyme activity, which is no longer used. The continuous monitoring method was adopted in the mid-1950s. This method is completed on an automatic biochemical analyzer, which can measure the initial speed of the enzyme reaction, and the result is far more accurate than the fixed time method, especially in high-concentration specimens. But this method is also affected by reaction time, reaction temperature, reagents, etc. (1) Timing method: (two-point method) A method to obtain the initial speed of the enzyme reaction by measuring the total change in the concentration of the product or enzyme substrate in a certain period of time (t1 to t2) after the start of the enzyme reaction. Among them, t1 often takes the time when the reaction starts. The enzyme and the substrate act at a certain temperature for a fixed period of time, and the reaction is completely stopped by adding strong acid, strong base, protein precipitation agent, etc. (also called the suspension reaction method). Add reagents to enter the chemical reaction and color to detect changes in substrates and products. The most basic point of this method is to measure the change of the substrate or substance after stopping the reaction. Advantages: Simple and easy to implement, not high requirements for reagents.rnDisadvantages: It is difficult to guarantee the authenticity of the measurement results. It is difficult to determine whether the enzymatic reaction is in the linear phase during the reaction period. As the incubation time continues, enzyme denaturation and deactivation accelerate. (2) Continuous monitoring method: also known as kinetic method or rate method, continuous reaction method. During the enzymatic reaction, the instrument is used to monitor the changes in the concentration of a certain reaction product or substrate over time, and the initial rate of the enzymatic reaction is calculated by calculation. The continuous monitoring method can select the substrate or product change rate in the linear phase to calculate the enzyme activity based on the continuously measured data. Therefore, the continuous monitoring method is more accurate than the timing method. In actual work, the enzyme coupling method using tool enzymes has become the most widely used and most frequent method for measuring the concentration of enzyme activity. (3) Equilibrium method: The method of calculating the enzyme activity by measuring the total change in the concentration of the product or substrate from the beginning of the enzyme reaction to the equilibrium of the reaction is also called the end point method. 2. Classification by detection method: (1) Spectrophotometry; (2) Polarization method; (3) Fluorescence method; (4) Electrochemical method; (5) Chemical reaction method; (6) Nuclide determination method; (7) Calorimetryrn