NADH fluorescence

Reisinger, C., van Assema, F., Schuermann, M. et al. (2006) A versatile colony assay based on NADH fluorescence. Journal of Molecular Catalysis B-Enzymatic, 39, 149-155. 

S. A. Siano and R. Mutharasan, NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes, Biotechnol. Bioeng. 37, 141-159 (1990). 

A. K. Srivastava and B. Volesky, NADH fluorescence in a carbon-limited fermentation, Biotechnol. Bioeng. 38, 191-195 (1991b). 

N. S. Wang and M. B. Simmons, Effect of background fluorophores on the NADH fluorescence probe signal, Biotechnol. Tech. 5, 241-246 (1991). 

S. C. W. Kwong, L. Randers, and G. Rao, Consistency evaluation of batch fermentations based on online NADH fluorescence, Biotech. Prog. 8, 410-412 (1992). 

Fluorescence may also be enhanced. Sometimes a compound has a low quantum yield in aqueous solution but a higher one in nonpolar media. The dyes tolu-idinyl- and anilinyl-naphthalene sulfonic acid fluoresce very weakly in water, but strongly when they are bound in the hydrophobic pockets of proteins. Interestingly enough, if they are bound next to a tryptophan residue, they may be excited by light that is absorbed by the tryptophan at 275 to 295 nm and whose energy is transferred to them. Tryptophan and NADH fluoresce relatively weakly in water, and their fluorescence may be enhanced in the nonpolar regions of proteins. 

Figure 20.17 (a) Proportion of fluorescence intensity decreased by 15nm nanospheres for ail samples. = 280 nm and =335 nm corresponding to protein fluorescence, (b) Proportion of total NADH fluorescence quenched by nanospheres, = 340 nm and miss 440 nm corresponding with NADH fluorescence. Cited from reference 52. 

Fluorescent band with emission maximum at 425 nm, distinct from albumin and NADH Fluorescence in whole serum before and after addition of CK assay mixture... 

Fig. 1. The activity of Na /K -ATPase is measured using an enzymatic essay and is proportional here to the decrease of NADH fluorescence intensity, If measured at 460 nm at selected pressures and 37 C [19].

In contrast, linear curves are obtained for the enhancement of NADH fluorescence when excited at 330 nm where no excitation transfer from the protein is possible. The tertiary structure provides a possible and simple explanation for these experiments in terms of the geometrical arrangement of the four tryptophans and the two active sites of the whole molecule as illustrated in Fig. 15. This interpretation is quite different from the structural implications of the geometric quenching discussed by Holbrook et ai. (199). 

The dihydronicotinamide ring of NADH fluoresces weakly at 470 nm when excited within its absorption band. The intensity of fluorescence in water is low and can be increased by solution in organic solvents 30) and shifted to shorter wavelengths (330 nm) by cleaving the pyrophosphate bond and thus removing the interaction with adenine (231). The fluorescence is increased on combination with the enzyme [122,2. but can be either enhanced or quenched when a ternary complex forms. 

The substrate rapidly forms a ternary complex with E-BH+ which has quenched NADH fluorescence. This process can be observed to occur before the spectrum of NADH is destroyed in the catalytic step. 

Ewly transient kinetic studies of pig H4 and M LDH are summarized by Gutfreund 291). Resolution of the intermediates in the LDH reaction has depended upon measurement of protein fluorescence, NADH fluorescence, NADH absorbance at 340 nm, and proton uptake with dyes 269). These characteristics are listed in Table XXI. 

Phase 1. An instantaneous (<1 msec) formation of 0.1-0.3 mole NADH per subunit. This instantaneously formed compound has absorbance at 340 nm, little NADH fluorescence, and quenched protein fluorescence. No proton is liberated. 

Phase 2. A first-order process in which all the remaining sites become saturated with NADH. During this process NADH fluorescence is enhanced but protein fluorescence remains quenched. Protons are liberated at the same rate as NADH is produced. 

Siidi 291a) has also examined the transient changes in NADH fluorescence during the forward reaction and has observed these three phases. 

Phase 1 [identified as step 1 in Eq. (7)] is complete within the mixing time and gives a compound retaining absorbance at 340 nm, but with quenched NADH and protein fluorescence. Phase 2 is a first-order process in which absorbance at 340 nm is destroyed, protein fluorescence appears, and NADH fluorescence remains quenched. If this first-order process were after the redox step [step 4 in Eq. (7) ], then the first turnover of NADH would be very fast. This is not observed. If the first-order process were the redox step itself [step 3 in Eq. (7)] then it would be slower with NADD than with NADH by a factor of 6 to 7, as with alcohol dehydrogenase (293). No appreciable isotope effect is measured. Thus the first-order phase must be identified with an isomerization of the ternary complex with NADH [step 2 in Eq. (7)] before the redox step (269,279). Siidi (293a) has also observed two phases in the reverse reaction and has deduced the on rate for pyruvate. The kinetics do not indicate the... 

The classical example of a biochemical oscillator is glycolysis. Damped oscillations were observed in the NADH fluorescence of yeast cell suspensions. Sustained oscillations were notable in yeast glycolysis (Figure 8.25) within a clearly defined range of substrate infusion rates, outside of which steady-state behavior was obtained. 

Optodes provided with non-fluorescent esters of fluorophores have been used for the determination of external enzyme activities. The fluorophores are liberated by the enzymes and then seen by the optical Ober [214], As ecamples of p(02)-modulated optical biosensors, a glucose probe [213] and an ethanol probe [216] can be mentioned sensors based on glucose, alcohol, and other oxidases were reviewed by Opitz and Lttbbers [217]. The advantages of these 02-dependent optical biosensors are that, unlike corresponding amperometric sensors, they do not consume O2 and that they are strictly diffusion limited in their response. Fiber-optical devices are also available for the determination of substrates of dehydrogenases the NADH fluorescence produced by the immobilized enzyme is measured as a function of time [218, 219]. 

The NAD+ chromophore absorbs at 260-270 nm and does not fluoresce. NADH, on the other hand, has an additional absorption band at 340 nm (Fig. 7.2.8) and a fluorescence band at 350 nm. NADH fluorescence has been used to follow NAD+-catalyzed reactions down to a detection limit of lO mol L . 

Using a pulsed N-2 laser combined with a fiber-optic probe and photomultipliers, the NADH fluorescence was measured in the brain cortex of rats. After intraperitoneal application of NADH (50mg/kg), an increase in the intensity of the cortical NADH fluorescence of about 18% was observed for approximately 30min compared with the fluorescence intensity in the control group. Neither NAD+ (the oxidized form of NADH) nor nicotinamide (both at concentrations of 50mg/kg) showed any effect on the NADH fluorescence in the cortex for the entire measurement period of 120min. 

Lactate Lactatedehydrogenase/nylon membrane + NADH Fluorescence... 

Figure 5.4 The reaction of pig heart lactate dehydrogenase with lactate and NAD. The four panels show (a) two phases of NADH formation (enzyme bound and free), (/>) NADH fluorescence, which is predominated by the enzyme-NADH complex after pyruvate dissociation, (c) protein fluorescence quenching monitoring concentration of all enzyme-NADH complexes and (d) phenol red absorbence monitoring two phases of proton liberation. (For detail see text and Whitaker ef n/., 1974.)...

---