Soluble dinucleotides such as nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide (NAD) can be reduced in a reversible way by adding a pair of hydrogen ions. In reversible reactions, these molecules mainly serve as coenzymes, but they also play a major role in other reactions.
NADP is typically involved in steroid synthesis, fatty acid synthesis, and other reductive synthesis reactions. During anaerobic metabolism, either dehydrogenase enzyme or the complex I of the electron transport chain reoxidizes the NADP molecule. NAD is often utilized as an acceptor of reducing equivalents in catabolism reactions, mainly b-oxidation of fatty acids, the tricarboxylic acid cycle, and glycolysis.
NAD is basically a multiple ringed structure (Figure 1) that characteristically goes through redox reactions within its nicotinamide ring. The NADP is a closely associated molecule and is phosphorylated on the 2’ position of the adenosine ribose ring.
Figure 1. Structure and redox reaction of nicotinamide adenine dinucleotide (NAD+). Figure (A) depicts the chemical structure of the nicotinamide adenine dinucleotide (NAD+) molecule, while (B) demonstrates the bi-directional redox-reaction between NAD and NADH that is catalyzed by dehydrogenase enzymes.
With respect to quantitation, NAD molecule or NADP molecule are involved in enzymatic dehydrogenase reactions that leverage the characteristic of NADPH/NADH, which incidentally are reduced forms of these molecules, to absorb light at 340 nm wavelength.
The oxidized forms, however, do not absorb light. In a similar way, when stimulated at 340 nm, the reduced forms create fluorescent emission at 445 nm, whereas the oxidized forms do not.
With the aid of these physical properties, investigators can easily determine the reactions that involve a transformation of NAD and NADP’s oxidative state. Figure 2A illustrates an example of this reaction, where in order to change lactate to pyruvate by lactate dehydrogenase (enzyme) the equimolar quantities of NAD+ have to be changed to NADH.
Dehydrogenases are not directly associated with the coenzymes. Excluding this enzyme, it is possible to determine the enzyme reactions by combining the enzymes’ product to a dehydrogenase. This dehydrogenase in turn utilizes that product to act as a substrate, as shown in Figure 2B.
Figure 2. Outline of two reactions using NAD+ as a coenzyme. Reaction A demonstrates a reaction directly linked with the reduction of the coenzyme NAD+. Lactate can be quantitated by formation of NADH as a result of the enzymatic conversion of lactate to pyruvate by the enzyme lactate dehydrogenase. Reaction B depicts a coupled reaction where ATP can be quantitated by a loss of NADH. One of the products of the initial reaction catalyzed by phosphoglycerate phosphokinase (PGK) is utilized as a substrate in a second reaction involving NADH that is catalyzed by glyceraldehyde phosphate dehydrogenase (GAPD).
In case there is surplus amount of dehydrogenases, the speed of disappearance or appearance of NADH could be utilized to determine an enzyme, where NADH is not used as a substrate.
Materials and Methods
For the experiment, Sigma Chemical Company supplied NAD+ and NADH vials that were pre-weighed. Corning provided solid black fluorescence microplates with catalogue number 3915, as well as half and regular area 96-well UV transparent microplates with catalogue numbers 3679 and 3636, in that order.
Then, utilizing TE pH 8.0 (10mM Tris, 1mM EDTA) as the buffer, 1mg/ml of stock solutions of individual compound were prepared. Subsequently, a Shimadzu UV-1700 spectrophotometer was used to validate the NADH solution concentrations through absorbance at 340 nm.
Then, utilizing TE, pH 8.0 as the diluent, additional dilutions were made and 200 µl aliquots of individual dilution were transferred into the microplate wells in four replicates. Next, the Synergy™ 2 Multi-Mode Microplate Reader was used to make absorbance measurements at 340 nm.
About 100 aliquots in half-area 96-well UV-transparent microplates, ranging from 200 to 600 nm in increments of 1nm, were used to perform absorbance spectral scans. Before plotting, the TE buffer-only absorbance was subtracted from the data.
The same microplate reader was used to measure the fluorescence of the entire compounds, using a combination of 440 nm, 40 nm bandwidth emission filter and 340 nm, 30 nm bandwidth excitation filter along with a 400 nm cut off dichroic mirror. Then, by means of the tungsten halogen light source, the information was obtained.
With the aid of spectral scans made from 200 to 600 nm of solutions of NADH and NAD+, the variations between the reduced forms and oxidized forms of the molecule were observed.
It was seen that NAD+ solution has almost no absorbance peak at 340 nm wavelength (Figure 3), while NADH solution has a sharp absorbance peak at this wavelength. This disparity was considered to track various assays. In the following experiments, an excitation and an absorbance wavelength of 340 nm were utilized.
Figure 3. Spectral Scans of NADH and NAD+ solutions. Aliquots (100 µl) of NADH (blue dots) and NAD+ (red dots) solutions (1 mg/ ml) were aliquoted into half area-UV transparent plates and a spectral scan from 200 nm to 600 nm in 1 nm increments performed. Data was plotted using Gen5™ Data Analysis Software.
For concentrations of NADH and NAD+ solutions spanning from 0 to 500 µg/ml (Figure 4), the absorbance was measured. In the case of NADH samples, the absorbance beyond this range amplified in a linear way, while in the NAD+ samples, there was almost no significant increase in absorbance.
Here, BioTek’s Gen5™ Data Analysis Software can be used to create a least means squared linear regression analysis, keeping 1.0 as a coefficient of determination (r2) value; 2% was the typical coefficient of variation (%CV) of the standards with maximum percent difference arising in the lower concentrations of NADH.
It was observed that under suitable conditions, NADH concentrations down to 975ng/ml were statistically dissimilar from the TE buffer-only control.
Figure 4. NADH and NAD+ concentration curve measured using absorbance at 340nm. Serial dilutions of NADH and NAD+ ranging from 0-500 µg/ml were made using TE pH 8.0 aqueous buffer as the diluent. The absorbance was determined using a Synergy™ 2 Multi-Mode Microplate Reader in absorbance mode. Gen5™ Data Analysis Software was used for reader control and data capture, as well as linear regression analysis of the data.
A hyperbolic response was seen when measuring the NADH fluorescence for concentrations spanning from 0 to 500 µg/ml (Figure 5), whereas when the oxidized form of NAD was excited by 340 nm light, it did not display any fluorescent emission at 440 nm.
A considerable amount of the excitatory light is absorbed by NADH when its concentration is relatively high. Since the NADH exhibits incomplete saturation of light at the wavelength of 340 nm, one can see a quenching of NADH fluorescence at higher concentrations of NADH solution.
Figure 5. NADH concentration curve measured using fluorescence. Serial dilutions of NADH and NAD+ ranging from 0-500 µg/ml were made using TE pH 8.0 aqueous buffer as the diluent. The fluorescence was determined using a Synergy™ 2. Gen5™ Data Analysis Software was used for reader control and data capture, as well as a 4-parameter logistic fit best fit of the data.
However, a 4-parameter logistic fit of the data showed excellent agreement between the fluorescence and concentration. Given that the coefficient of determination (r2) value was measured to be 0.999, unknown concentrations in this range can be easily established. A linear response is observed when lower concentrations of NADH solutions, i.e. 0 to 12.5 µg/ml, are determined (Figure 6).
Figure 6. Linearity of low NADH concentrations. Dilutions of NADH ranging from 0-12.5 µg/ml were made and their fluorescence determined. Gen5™ Data Analysis Software was used for reader control and data capture, as well as sample blanking and linear regression analysis.
Taking the linear regression analysis of the resultant data, it is considered that a straight-line function optimally describes the link between the fluorescence and the NADH concentration at low concentrations.
Under suitable sensitivity conditions, 37 ng/ml of NADH concentrations was statistically dissimilar from the TE-only control, as shown in Figure 6. Considering the sample volume in a well, this limit of detection indicates the measurement of 1.03 x10-11 moles of NADH.
As a rule, changes in absorbance at 340 nm helped in quantitating enzymatic reactions where NADH was also involved. The coenzymes in reduced form were fluorescent upon stimulating with 340 nm light.
This article shows how the same instrument can be used to perform both techniques of quantitating the NADH in solution. As predicted, when fluorescence is used, low concentrations of NADH are easily detected.
The photomultiplier amplifies small amounts of light signal, allowing them to be identified against a low background. However, the reverse is true when absorbance is used to determine the low levels of compounds.
This is due to the fact that only a small amount of light signal is taken in against a high background. With regard to limits of detection, using fluorescence to measure the concentrations of NADH and NADPH leads to better sensitivity than that of absorbance.
This article has shown how the Synergy™ 2 Multi-Mode Microplate Reader can be effectively used to track changes in the levels of NADH solutions via fluorescence or absorbance modes. It also describes how different dilutions of NADH can be determined and how phosphorylated relative NADPH shows analogous properties.
With the aid of these measurements, different enzymatic assays using these coenzymes for transfer of hydrogen ions can be determined. However, this can be done if there is a known molar association between the target analyte and the destruction or creation of NADPH or NADH.
There are some assays, which instead of using an endpoint determination, utilize the reaction kinetics of the decrease or increase in the quantities of the reduced form of coenzymes. While this article does not show the application of kinetic reactions, the Synergy™ 2 Multi-Mode Microplate Reader in tandem with the Gen5™ Data Analysis Software can make the kinetic determinations and can also measure the reaction rate.
Produced from materials originally authored by Paul Held, Ph.D, Senior Scientist, Applications Department, BioTek Instruments, Inc.
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