Glutathione reductase (GSH) is a key enzyme to maintain the REDOX balance of cells, and its activity is widely used in biomedicine to assess the antioxidant capacity and oxidative stress level. NADPH colorimetric method was the main detection method, and new techniques such as fluorescence method and HPLC improved the detection sensitivity and specificity.
What is glutathione reductase?
Glutathione Reductase (GR) is one of the important enzymes to maintain the REDOX balance in the cell. It catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). Reduced glutathione is an important antioxidant in the body and is involved in the antioxidant defense system of cells. GR activity detection is often used to assess the antioxidant capacity and oxidative stress level of cells or tissues, and is widely used in biomedicine, toxicology, pharmacology and environmental science.
Biological function of glutathione reductase (GR)
GR plays a key role in the body's antioxidant system. GSH is the main antioxidant in the reduction of peroxides by glutathione peroxidase (GPx), during which GSH is oxidized to GSSG.
GR maintains the intracellular GSH/GSSG ratio by reducing GSSG back to GSH, thereby maintaining the intracellular reduction environment. The activity of this enzyme is essential to prevent oxidative damage to cells.
Principles of GR detection
The basic principle of GR detection is to evaluate the GR activity indirectly by measuring the oxidation rate of NADPH during the reaction.
During the reduction of GSSG to GSH catalyzed by GR, NADPH is oxidized to NADP+^++.
By measuring the absorbance change of NADPH at 340 nm and calculating its consumption rate, the activity of GR was calculated.
Common methods of GR detection
NADPH colorimetry:
This method is the most classical and commonly used GR detection method, based on the characteristic absorbance of NADPH in the reduced state.
By measuring the absorbance change of NADPH at 340 nm, the rate of GR catalytic reaction can be directly reflected.
Experimental steps:
- A reaction system containing oxidized glutathione (GSSG), NADPH and an appropriate buffer was formulated.
- GR was added to the sample to initiate the reaction.
- Using a spectrophotometer, changes in NADPH absorbance are monitored in real time at 340 nm wavelengths.
- GR activity was calculated based on the rate of absorbance decline over time.
Fluorescence method:
The fluorescence method detects changes in concentration by using NADPH or NADP+^++ specific fluorescent probes.
The method has high sensitivity and is suitable for detecting GR activity in samples with low concentration.
Experimental steps:
- Preparation of fluorescence probe reaction system with NADPH.
- GR samples were added to start the reaction.
- Changes in fluorescence signal intensity are detected at specific excitation and emission wavelengths.
- The GR activity was calculated by the rate of change of the fluorescence signal.
High Performance Liquid Chromatography (HPLC) :
The activity of GR was determined indirectly by HPLC by separating and quantifying the contents of GSSG and GSH. The method has high accuracy, but the operation is relatively complicated, and it is usually used for fine research.
Experimental steps:
- The reaction system is formulated with GSSG, NADPH and buffer.
- GR samples were added and sampled after reaction for a period of time.
- The concentrations of GSH and GSSG were detected by HPLC.
- The activity of GR was estimated by the changes of GSSG and GSH before and after the reaction.
Oxygen electrode method:
This method indirectly assays GR activity by detecting oxygen consumption during NADPH oxidation and is suitable for precise enzyme kinetics studies.
Experimental steps:
- Prepare a reaction system containing GSSG and NADPH.
- GR samples were added to the oxygen electrode apparatus to initiate the reaction.
- Real-time monitoring of oxygen concentration changes in the reaction system.
- The activity of GR was estimated by oxygen consumption rate.
Experimental procedure of GR detection
Taking the classic NADPH colorimetric method as an example, the experimental steps of GR detection are as follows:
Sample preparation:
The sample may be cell homogenate, tissue extract or purified enzyme solution. Long exposure to air should be avoided when handling samples to prevent oxidation of GSH.
Reaction system preparation:
In the appropriate buffer (usually phosphoric acid buffer or Tris-HCl buffer), NADPH and GSSG are added to form the reaction system. The proper concentration of NADPH and GSSG can be determined by a pre-established standard curve.
Sample addition and starting reaction:
GR in the test sample was added to the reaction system and quickly mixed to start the reaction. Ensure that reaction conditions such as temperature, pH, etc. are appropriate to ensure optimal enzyme activity.
Real-time detection:
The absorbance of the reaction system was continuously monitored at 340 nm wavelength using a spectrophotometer. Data is usually recorded every few seconds or minutes until NADPH is exhausted or equilibrium is reached.
Result calculation:
According to the NADPH consumption rate, the enzyme activity of GR was calculated by the formula. Enzyme activity is usually expressed as the amount of NADPH consumed per milligram of protein per unit time.
GR detection application
GR detection has a wide range of applications in a variety of research areas:
Oxidative stress studies:
GR is a key enzyme in the body's antioxidant defense system, and its activity changes can reflect the oxidative stress state of cells or tissues. In the study of oxidative stress-related diseases such as cancer, diabetes, neurodegenerative diseases, GR activity detection is of great significance.
Antioxidant screening:
In the development and evaluation of antioxidants, GR activity detection is one of the commonly used indicators. By measuring the effect of antioxidants on GR activity, it is possible to evaluate the enhancement effect of antioxidants on cellular antioxidant capacity.
Environmental Toxicology studies:
GR activity can also be used as a biomarker to assess the toxic effects of environmental pollutants (such as heavy metals, pesticides, etc.) on organisms. Many environmental poisons can trigger oxidative stress, and the potential harm of pollutants to organisms can be assessed by detecting changes in GR activity.
Metabolic Disease Research:
In many metabolic disorders and aging-related diseases, GR activity changes are closely related to pathological processes. GR detection has important application value in studying the pathogenesis of these diseases and the effect of drug intervention.
Pharmacological studies:
GR testing is also used to evaluate the antioxidant effects and toxicity of drugs. In the process of drug development, the change of GR activity can be used as an evaluation index of drug effect, especially in the development of antioxidant drugs.
conclusion
Glutathione reductase (GR) is a key enzyme in the maintenance of cellular REDOX balance, and its activity detection is of great significance for the study of antioxidant system, oxidative stress and related diseases.
The classical NADPH colorimetric method has become the main method for GR detection because of its simple operation and high sensitivity.
With the development of detection technology, the application of new methods such as fluorescence and HPLC has further improved the sensitivity and specificity of detection, providing more diversified means for the study of GR activity.
