Why HEPES Buffer 7365-45-9 Is Used In Lactate Dehydrogenase Detection

Lactate dehydrogenase (LDH), as one of the key enzymes in the intracellular glycolysis pathway, undergoes changes in its activity under various pathological conditions. Accurately measuring LDH activity is not only of great significance for basic scientific research, but also an important tool for clinical diagnosis. Choosing the appropriate buffer is one of the key factors in ensuring the accuracy of the test results during this process.

4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES buffer), as a widely used biological buffer, is gradually becoming the core component of lactate dehydrogenase detection kits. This article will analyze in detail from multiple perspectives why HEPES buffer is used in lactate dehydrogenase detection.

HEPES buffer

1, Basic characteristics of HEPES

HEPES is a zwitterionic buffer containing amino and carboxyl groups, with the molecular formula C8H18N2O4S. It is a weakly alkaline, non-toxic, and odorless solid with good water solubility and chemical stability.

The buffering range of HEPES is between pH 6.8 and 8.2, and for the detection of LDH, its catalytic environment is usually within this pH range. Therefore, HEPES can effectively maintain the stability of reaction conditions and ensure the accuracy of enzyme activity measurement. In addition, the buffering capacity of HEPES is not affected by CO2 concentration, which is particularly important for cell culture and biochemical reactions in open environments.

2, Application of HEPES in LDH detection

HEPES, as one of the core components in the lactate dehydrogenase detection kit, plays multiple roles. The detection kit usually adopts a dual reagent design, with reagent I containing biological buffer HEPES, metal ion complex, LDH reaction substrate, surfactant, LDH enzyme activator, preservative, and water; Reagent II consists of HEPES, nicotinamide adenine dinucleotide oxidized NAD+, surfactant, preservative, and water. By adding different functional components separately, the reaction conditions can be better controlled and the interference caused by the turbidity of the sample itself can be reduced.

3, Improve detection accuracy and efficiency

Good stability: The buffering effect provided by HEPES ensures pH stability throughout the entire detection process, which is crucial for enzyme activity measurement.

High accuracy: By optimizing the formula and implementing strict quality control measures, consistent results are ensured for each test.

Wide linear testing range: able to cover a wide range of LDH activity levels, adapt to different sample types, and meet different clinical needs.

Strong anti-interference ability: effectively resists the influence of external factors such as temperature changes or sample impurities, ensuring the authenticity of data.

No need for pre dilution: simplifies experimental steps, shortens detection time, especially suitable for busy clinical environments.

4, Reaction principle and measurement

The process of LDH catalyzed reduction of pyruvic acid to L-lactic acid is a reversible reaction, and when the concentration of lactic acid is high, it promotes the reverse reaction, that is, the synthesis of pyruvic acid from lactic acid. Under suitable pH conditions maintained in HEPES buffer, L-lactic acid reacts with NAD+to produce pyruvic acid and NADH. NADH exhibits characteristic absorbance at a wavelength of 340nm, which means that LDH activity can be quantified by monitoring changes in absorbance.

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