What Is NADH Absorbance and Why Is It Important?

Nicotinamide adenine dinucleotide (NADH) is a coenzyme that acts as an electron carrier, shuttling energy within cells. It is part of metabolic processes like glycolysis and the citric acid cycle, facilitating the breakdown of nutrients. This molecule exists in two states: a reduced form, NADH, which carries electrons, and an oxidized form, NAD+, which has donated them. The ability to switch between states is central to energy production, and a unique physical characteristic of NADH allows scientists to track this activity.

The Principle of NADH Absorbance

Light absorbance provides a way to measure biological processes. A spectrophotometer is an instrument that measures the amount of light absorbed by a sample by passing a beam of a specific wavelength through it. The instrument then detects how much light emerges on the other side, and the difference reveals the substance’s absorbance.

This principle is useful for studying NADH because the reduced form strongly absorbs ultraviolet (UV) light at a wavelength of 340 nanometers (nm). In contrast, its oxidized counterpart, NAD+, does not absorb light at this wavelength. This difference in their properties allows scientists to distinguish between the two forms.

The reason for this difference lies in the chemical structure of the molecule’s nicotinamide ring. When NAD+ accepts electrons to become NADH, the arrangement of bonds within this ring changes. This alteration affects how the molecule interacts with UV light, creating the absorbance peak at 340 nm. This phenomenon makes the conversion between NAD+ and NADH an observable event.

Measuring Enzyme Activity with NADH

The absorbance property of NADH is used to measure the speed of enzymatic reactions. By tracking the change in absorbance at 340 nm, scientists can monitor an enzyme’s rate. This technique, called a coupled enzyme assay, links the enzyme’s activity to the production or consumption of NADH.

For example, in a reaction where an enzyme reduces NAD+ to NADH, the concentration of NADH will rise. A spectrophotometer will detect a corresponding increase in light absorbance at 340 nm over time. The faster the absorbance increases, the faster the enzyme is working.

Conversely, many reactions consume NADH. The enzyme lactate dehydrogenase (LDH), active during anaerobic respiration, converts pyruvate to lactate. This reaction requires the oxidation of NADH back to NAD+. By monitoring a sample, scientists can observe a steady decrease in absorbance at 340 nm as NADH is used, directly measuring the enzyme’s activity.

Calculating Reaction Rates

To calculate a precise reaction rate from absorbance changes, scientists use the Beer-Lambert Law, an equation in spectrophotometry expressed as A = εcl. In this formula, ‘A’ is the absorbance value from the spectrophotometer. This value is directly proportional to the concentration of the substance being measured.

In the formula, ‘c’ stands for the concentration of NADH, and ‘l’ represents the path length, which is the distance light travels through the sample. In most spectrophotometers, this is standardized using cuvettes that have a path length of 1 centimeter. This standardization simplifies calculations and ensures consistency.

The final component, ‘ε’ (epsilon), is the molar extinction coefficient, a constant defining how strongly a substance absorbs light at a specific wavelength. For NADH at 340 nm, this value is 6,220 M⁻¹cm⁻¹. With this constant, scientists can use the change in absorbance over time to calculate the exact rate of NADH production or consumption, reflecting the enzyme’s activity.

Practical Applications in Science and Medicine

The measurement of NADH absorbance has applications in research and medical diagnostics. In clinical settings, it is used to assess patient health by measuring the activity of enzymes in blood samples. For instance, elevated levels of enzymes like alanine aminotransferase (ALT) and aspartate aminotransferase (AST) can indicate liver damage. Assays for these enzymes are coupled to NADH oxidation, where a rapid decrease in absorbance at 340 nm suggests high enzyme activity and potential tissue injury.

In metabolic research, NADH-based assays help map biochemical pathways. Scientists use this technique to study how factors like disease, diet, or exercise impact cellular metabolism. By isolating mitochondria, researchers can measure NADH production to understand how efficiently cells generate energy and how metabolic disorders might disrupt this process.

This method is also used in the pharmaceutical industry for drug discovery. High-throughput screening platforms use NADH-coupled assays to test thousands of potential drug compounds. For example, a company might screen for compounds that inhibit HMG-CoA reductase, an enzyme in the cholesterol synthesis pathway. Because this reaction involves a related molecule, NADPH, a similar absorbance-based assay can identify drug candidates that slow the reaction rate.