Resorufin Anion Research Articles

Excited‐State Hydrogen Bonding Strengthening and Weakening with Intramolecular Charge Transfer in Resorufin‐Water Complexes: A TD‐DFT Study

AbstractThe geometric structures, infrared spectra and hydrogen bond binding energies of the various hydrogen‐bonded Res−‐water complexes in states S0 and S1 have been calculated using the density functional theory (DFT) and time‐dependent density functional theory (TD‐DFT) methods, respectively. Based on the changes of the hydrogen bond lengths and binding energies as well as the spectral shifts of the vibrational mode of the hydroxyl groups, it is demonstrated that hydrogen bonds HB‐II, HB‐III and HB‐IV are strengthened while hydrogen bond HB‐I is weakened in the four singly hydrogen‐bonded Res−‐Water complexes upon photoexcitation. When the four hydrogen bonds are formed simultaneously between one resorufin anion and four water molecules in the Res−‐4Water complex, all the hydrogen bonds are weakened in both the ground and excited states compared with those in the corresponding singly hydrogen‐bonded Res−‐Water complexes. Furthermore, in complex Res−‐4Water, hydrogen bonds HB‐II and HB‐IV are strengthened while hydrogen bonds HB‐I and HB‐III are weakened after the electronic excitation. The hydrogen bond strengthening and weakening in the various hydrogen‐bonded Res−‐water complexes should be due to the redistribution of the charges among the four heteroatoms (O1‐3 and N1) within the resorufin molecule upon the optical excitation.

A DFT/TDDFT study of hydrogen bonding interactions between resorufin anion and water molecules in the excited state

In order to explore the hydrogen bonding interactions between resorufin anion (denoted as Res − in this paper) and water molecules, we have calculated the geometric structures as well as the total energies of the various hydrogen-bonded Res −–water complexes composed based on the atomic charges obtained from the NBO analysis. We found that there are four sites (O 1–3, N 1) within the Res − molecule through which intermolecular hydrogen bonds can be formed with water molecule. Furthermore, by comparing the hydrogen bond lengths and the relative energies of the four singly hydrogen-bonded complexes Res −–H 2O, we found that hydrogen bonds formed at the two carbonyl oxygen atoms (O 2 and O 3) are stronger than those formed at heteroatom O 1 and N 1 which can be reasonably predicted by the highest negative charge localized on the two former atoms. Moreover, as the number of the water molecules hydrogen-bonded to the Res − increases, the hydrogen-bonded complex Res −–water becomes more stable although the strength of each hydrogen bond decreases. In addition, the electronic spectra of the various hydrogen-bonded Res −–water complexes as well as the Res − monomer are also calculated. We found that, compared with that of the Res − monomer, the excitation spectra of the hydrogen-bonded Res −–water complexes are mostly blue-shifted in the S 2 and S 3 states while they are all red-shifted in the higher excited states, especially in states S 10, S 11 and S 12. According to the relationship between electronic spectral shifts and electronic excited-state hydrogen-bonding dynamics first clarified by Zhao and coworkers [51], we demonstrate that, for all the hydrogen-bonded Res −–water complexes, most of the hydrogen bonds are weakened in the S 2 and S 3 states while they are all strengthened in excited states S 10, S 11 and S 12.

Effects of Electrolyte Concentration on the Rotational Dynamics of Resorufin

We report on the rotational diffusion dynamics of the anionic chromophore resorufin in water and N-octyl-2-pyrrolidone (NOP) solutions as a function of solution electrolyte concentration. Our data show that resorufin exhibits a single exponential anisotropy decay in aqueous solutions containing up to 0.1 M lithium perchlorate (LiClO(4)). In contrast to the observed behavior of resorufin in pure NOP, where biexponential decay occurs, we also observe a single exponential anisotropy decay for resorufin in NOP with the addition of up to 0.1 M tetrabutylammonium bromide (TBAB) or tetraoctylammonium bromide (TOAB). For resorufin in NOP, the reorientation time constant increases with increasing electrolyte concentration, consistent with complexation between the resorufin anion and the electrolyte ammonium cation. We observe a qualitatively different trend in the aqueous resorufin solutions and understand these data for both solvent systems in the context of interactions between the chromophore and cationic species present.

The effect of quercetin, a widely distributed flavonoid in food and drink, on cytosolic aldehyde dehydrogenase: a comparison with the effect of diethylstilboestrol

Quercetin is a flavonoid found in red wine and many other dietary sources. Observations concerning the state of ionisation and the stability of the compound over a range of pH are presented. Quercetin is a potent inhibitor of cytosolic aldehyde dehydrogenase at physiological pH when the concentration of either the substrate or the cofactor is relatively low, but it has an activatory effect when the concentrations of substrate and cofactor are both high (1 mM). Gel filtration experiments show that quercetin binds very tightly to the enzyme under conditions where the compound is neutral and when it is ionised. The binding is less in the presence of NAD +. Quercetin cuts down the ability of the resorufin anion to bind to the enzyme. The observations are explained by a model in which quercetin binds competitively to both the coenzyme-binding site and the aldehyde-binding site; binding in the latter location, when the enzyme is in the form of the E-NADH complex, accounts for the activation. The effects of quercetin are significantly different in some respects from those of diethylstilboestrol; this is explained by the latter being able to bind to the aldehyde site but not the NAD + site. The possibility that quercetin may affect aldehyde dehydrogenase in vivo is discussed.

Covalent modification of sheep liver cytosolic aldehyde dehydrogenase by the oxidative addition of coloured phenoxazine, phenothiazine and phenazine derivatives.

Aldehyde dehydrogenase acts as an esterase towards reactive esters such as p-nitrophenyl acetate, and although the subject of prolonged debate, it is now generally believed that the dehydrogenase and esterase actions of the enzyme involve the same active site and catalytic groups (see Kitson and Kitson, 1996, and references therein). With p -nitrophenyl dimethylcarbamate, the reactivity (compared to the acetate) is so drastically reduced that it takes several hours for this ‘substrate’ to acylate cytosolic aldehyde dehydrogenase (ALDH-l), and the rate of subsequent deacylation is essentially zero (Kitson et al., 1991). Thus the carbamate acts as an active-site-directed irreversible inactivator of the enzyme. It was thought that resorufin dimethylcarbamate (see Figure 1) would react likewise, but since the molar absorptivity of the resorufin anion (69,700) is so much greater than that of p-nitrophenoxide (18,320) (Kitson, 1996), the resorufin carbamate would be a much more sensitive active site titrant than the p-nitrophenyl compound. The results reported and discussed below show that this expectation was not borne out. Resorufin dimethylcarbamate does inactivate ALDH-I, but the chemistry of the reaction is more complicated and interesting than the simple acylation process expected from the previous work with the p-nitrophenyl equivalent; it may be termed ‘oxidative addition’.KeywordsMethylene BlueEthyl EtherCovalent ModificationOxidative AdditionSubstitution MechanismThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Covalent Modification Reactions Involving Cytosolic Aldehyde Dehydrogenase and a Variety of Resorufin Derivatives

The bromoacetates of resorufin and ofp-nitrophenol act simultaneously as very rapidly hydrolyzed substrates and as potent irreversible inactivators of cytosolic aldehyde dehydrogenase. Following inactivation, the resorufin orp-nitrophenoxide moiety is released fairly quickly by hydrolysis from the modified enzyme. Resorufin dimethylcarbamate and resorufin methanesulphonate inactivate aldehyde dehydrogenase in an “oxidative addition” reaction that is consistent with the involvement of the enzyme's catalytically essential cysteine residue with the quinonimine ring of the resorufin structure. The resulting covalently modified enzyme is yellow-orange and the redox properties of the colored label can be conveniently observed. Resorufin ethyl ether also slowly inactivates aldehyde dehydrogenase, but the resulting labeled enzyme is a surprising pink-violet color at pH values of about 5 to 10. The expected yellow-orange derivative is obtained at pH less than 4 or upon denaturation of the protein. The possible chemical identity of the species responsible for the pink-violet color is discussed. The resorufin anion binds noncovalently to aldehyde dehydrogenase apparently at the cofactor binding site, as it may be displaced by NAD+or NADH.

Resorufin in the Channels of Zeolite L

Zeolite L containing the resorufin anion (Res-) in its anionic framework can be prepared by incorporating the neutral resorufin molecule (ResH) from the gas phase and then exchanging the protons with potassium ions which leads to dark violet colored microcrystals. The reversibility of the deprotonation/protonation reaction of resorufin can be monitored by measuring the UV/vis absorption spectrum of the samples. The absorption spectra of Res- and ResH and their solvent dependence was interpreted using the EHMO-EDiT theory. While Res- is strongly fluorescent in solution, the fluorescence is completely quenched when Res- is located inside the channels of potassium zeolite L. This allows the investigation of the exit kinetics of the resorufin molecules as a function of the size of the solvent molecules which enter the zeolite channels and displace the incorporated resorufin molecules irreversibly. The following series was found for the displacement rate: water ≫ methanol > ethanol > 1-propanol ≳ 1-butanol. T...

Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450 scc

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450 scc (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F 1) second (F 2) and third (F 3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450 scc generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F 1 than in samples from F 2 or F 3. In cells, LH added simultaneously with the P-450 scc substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2–3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450 scc substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450 scc substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorescence for the substrate P-450 scc enzyme. In granulosa cells that were preloaded with the P-450 scc substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.

SINGLE CELL ENDOCRINOLOGY: ANALYSIS OF P-450scc ACTIVITY BY FLUORESCENCE DETECTION METHODS

A mechanism-based, fluorogenic probe for the cytochrome P-450scc (cholesterol side chain cleavage) enzyme, rate-limiting for the conversion of cholesterol to steroid hormones, is introduced and its application to the study of enzyme activity and regulation in single steroidogenic cells by several fluorescence detection methods is demonstrated. Reaction of the probe with P-450scc gives pregnenolone and the highly fluorescent resorufin anion. Spectroscopic changes in probe fluorescence, indicative of P-450scc activity, were monitored by steady-state fluorescence spectroscopy, flow cytometry, and microspectrofluorometry. This unique probe provides a nonradiometric indicator for real-time measurement of P-450scc activity in single living cells.

A spectroscopic investigation of the temperature and solvent sensitivities of resorufin

The electronic absorption spectra, fluorescence spectra and fluorescence lifetimes of resorufin anion in protic (ethanol–methanol 4 : 1 v/v) and aprotic (propiononitrile–butyronitrile 4 : 5 v/v) solvents in the temperature range 87–293 K are reported. Interaction of the resorufin probe with protic solvent results in the appearance of a spectroscopically identified species, not detected at room temperature. The resemblance of such a species to the neutral form of resorufin, as detected in water at pH < 5, indicates the involvement of a strong hydrogen bond between the solvent and the alkoxide ion of resorufin. Formation of such a complex is consistent with ‘super-stick’ behaviour of resorufin in alcohols. At temperatures < 150 K, where both protic and aprotic solvents are rigid, quenching of the emitting state of the resorufin anion is seen. Analysis of the lifetime behaviour vs. temperature provides the activation energy, ΔEs, for the solvent rearrangement affecting the emission in the 110–160 K temperature interval (ΔEs= 1980 and 3600 cm–1 for alcohol and nitrile solvents, respectively). These results are discussed in terms of ion–solvent interaction and of shielding of the anionic charge by primary solvation in alcohols.