Steady-state Fluorescence Emission Spectra Research Articles

Synthesis of Flavanol-4-ol and its Spectroscopic Properties in Aqueous Solution

by bubbling high purity argon (99.999%) through. The compound was then dissolved in water using double distilled water, which was obtained by passing distilled water through a Barnstead (U.S.A.) Nanopure II deionization system. The pH of the solution was adjusted by adding HClO4, NaOH or ammonium chloride buffer solutions which were prepared from air-free distilled water. These buffer solutions did not absorb any UV light with wavelength longer than 240 nm and therefore would not overlap with the characteristic peaks of flavan-4-ol. The UV/vis absorption spectra of the solutions were then taken by a UV/vis spectrophotometer (Uvikon, model 943, Italy) at room temperature. The steady-state fluorescence emission spectra were obtained on a Varian Cary Eclipse spectrofluorometer with 5 nm slits at room temperature after eliminating the oxygen from the solution by means of bubbling high purity argon (99.999%) through. To improve the accuracy, each sample was scanned 10 times and the average of these measurements was recorded as the emission spectrum.

Enhanced stokes shift of S 2 → S 0 emission and structural investigations of Sn(IV)Porphyrins doped hybrid borate glasses

In the present study, we report our results on Sn(IV)TPP and its derivatives doped hybrid borate glasses. The photophysical and structure of Sn(IV)Porphyrins in borate glass matrix was studied through micro-Raman, optical absorption, steady state and time resolved fluorescence emission techniques. The optical absorption spectrum shows red shift in Soret band and change in the intensity of Q-bands. Upon exposure to UV light at 260 nm, the glass samples exhibit strong blue emission and red emission at 365 nm. Steady state fluorescence emission spectrum of hybrid glasses exhibits dual emission bands originating from the S 2 → S 0 and S 1 → S 0 states. The appearance of large stokes shift (Δ υ), enhanced S 2 → S 0 emission at ∼482 nm and excitation dependent of S 1 → S 0 emission indicates that the structure of Sn(IV)Porphyrins were inherently modified in the borate glass matrix. Time resolved fluorescence of S 2 → S 0 emission was found to be in the range of 0.23–0.36 ns, S 1 → S 0 emission shows 1.23–1.57 ns and 9.12–9.63 ns, respectively. The results obtained in glass matrix were correlated with solution medium and the possible structures were investigated.

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

Characterization of N-vinyl carbazole/vinyl 4- tert-butyl-benzoate copolymers of several molar compositions: SEC, DSC and intramolecular excimers in dilute solutions

Several N-vinylcarbazole/vinyl p- tert-butyl-benzoate copolymers of different molar monomer compositions and their respective homopolymers have been synthesized by free-radical polymerization. Their solution properties were characterized by size-exclusion chromatography (SEC) coupled with a differential refractive index (RI) and multiangle light scattering (MALS). Differential scanning calorimetry (DSC) was also used to investigate the glass transition temperatures, T g. In addition, steady-state fluorescence emission spectra in a dilute solution of several fluid and non-fluid solvents were obtained to study the influence of monomer composition on the intramolecular carbazole excimer formation. The amount of excimers strongly depends on the solvent nature and copolymer composition. Molecular Dynamics (MD) simulations on iso- and syndiotactic homopolymer and copolymer fragments were used to obtain the probability of the conformations that satisfy excimer requirements. The analysis of the MD trajectories allows us to evaluate the amount and types of intramolecular excimers for the different systems.

Benzo[b]Fluorenone as a Quadruplex Interactive Agent (Qia): Binding Studies and Quadruplex Formation with the Human Telomeric HT4 Sequence

Many previously investigated agents capable of binding to and stabilizing G-quadruplex DNA conformations possess aromatic, planar chemical structures. Here, initial investigations on the interaction of benzo[b]fluorenone (BF) with the human telomeric forming oligonucleotide HT4 using circular dichroism (CD), thermal melting and steady-state fluorescence are reported. CD studies suggest that BF exhibits binding selectivity for G-quadruplex over model double-stranded DNA structures. BF binds to hybrid-mixed type quadruplex structures in the presence of physiological concentrations of K+ ions (100mM). However, even in the absence of salt, BF can induce an anti-parallel type quadruplex conformation. Thermal melting studies on quadruplex HT4 in the presence and absence of salt show BF has little effect on the melting temperature, suggesting that BF may associate with the G-quadruplex structure through a non-intercalating mode. As BF is known to be a solvatochromic fluorophore, steady state fluorescence emission spectra were taken for BF titrated with the HT4 quadruplex formed at 100mM K+, to explore the ability of BF to act as a potential probe of the environment of its binding site. Based on the red shift of BF emission, BF/quadruplex association appears to involve a relatively polar and hydrogen-bonded environment. This work was supported by NIH SCORE Grant S06 GM 060654.

Novel selenoesters fluorescent liquid crystalline exhibiting a rich phase polymorphism

A simple and efficient procedure for the synthesis of a new class of selenoesters 4a and 4b was developed. Polarized-light optical microscopy (POM), differential scanning calorimetry (DSC), and X-ray diffraction showed that the selenoester 4a with a shorter alkyl chain displayed a wide nematic range (ΔT = 110.7 °C), while 4b with a longer alkyl chain possesses a rich phase polymorphism: Cr → SmI → SmC → N → I mesophase sequence during heating cycle and I → N → SmC → SmI → SmX → Cr transition on cooling. For 4b, a large temperature range (135.7 °C) from crystal to isotropic phase can be observed. In addition, UV-Vis, steady-state fluorescence emission and excitation spectra in solution were also applied in order to characterize their photophysical behaviour. Compounds 4a and 4b are fluorescent in the blue region and present a Stokes shift at around 80 nm and 39 nm in dichloromethane and dioxane, respectively.

Fluorescence spectral resolution of tryptophan residues in bovine and human serum albumins

Static quenching and time-resolved emission spectra of tryptophan residues of BSA (2 Trp residues) and HSA (1 Trp residue) were performed in the presence of high concentrations of calcofluor white, a fluorophore that is specific to both carbohydrate residues and to hydrophobic sites in proteins. In the absence of calcofluor white, BSA and HSA emit with a maximum at 340 and 330 nm, respectively. Also, tryptophan residues in both proteins fluoresce with three identical lifetimes. Time-resolved spectra of HSA show that the three lifetimes emit at a maximum equal to 330 nm while spectra obtained from BSA show different peak positions for the three lifetimes. At high calcofluor concentrations, steady-state fluorescence emission spectrum of BSA displays a maximum at 330 nm instead of 340 nm in the absence of calcofluor. Fluorescence excitation spectra of the protein recorded in the absence and presence of calcofluor indicate the absence of protein conformational modification upon calcofluor white binding. Time-resolved emission spectra of the three lifetimes show identical peaks equal to 330 nm. Steady-state and time-resolved emission spectra performed on HSA in the presence of calcofluor do not show any modification in the emission peak (330 nm) indicating the absence of any conformational change and confirming the fact that the shift observed for tryptophan residues emission in BSA is the result of fluorescence quenching of Trp-134 residue.

Analysis of fluorescence spectra of PRODAN in protic solvents as the superposition of two Gaussian functions

The steady-state fluorescence emission spectra of 6-propionyl-2-dimethylaminonaphthalene (PRODAN) in aqueous solutions of methanol, ethanol and 1-propanol were studied in a wide range of concentrations. The flourescence of PRODAN was also studied in aqueous solutions (from 3 × 10−3 to 21 × 10−3 mole L−1) of dodecyltrimethylammonium bromide (DTAB), a micelle-forming ionic surfactant. In all cases, experimental data, as a function of wavelength, are very well fitted by the sum of two Gaussian functions. In alcohol solutions, the maximum of lower wavelength depends linearly both on concentration and polarizability. In DTAB solutions, the maximum of higher wavelength varies smoothly with concentration, but the lower wavelength maximum changes abruptly at the Critical Micellar Concentration. Results are explained by assuming that the fluorescence emission of PRODAN is originated in transitions from two excited states which are close in energy but have dissimilar interactions with the probe environment.

Nile Red Is a Fluorescent Allosteric Substrate of Cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) plays a critical role in the metabolism of many drugs. CYP3A4 exhibits extraordinary substrate promiscuity and unusual allosteric kinetics. In addition, many CYPs catalyze sequential oxidations on a single substrate, but in most cases, mechanistic details of these processes are not well-established. As a result, in vivo clearance of many drugs and their metabolites is difficult to predict on the basis of the complex in vitro kinetics, and new in vitro probes are required to understand these behaviors. The near-IR fluorescent probe Nile Red, which has strong solvatochromic behavior, was investigated as a probe of allostery and sequential metabolism with CYP3A4. Nile Red binds with apparent Kd values of 0.05 and 2.3 muM, based on a sigmoidal dependence of heme spin state on Nile Red concentration, where the first equivalent of Nile Red increased the high-spin fraction by only 13% of the total change at saturation. Mass spectrometry analysis indicates that Nile Red is metabolized sequentially by CYP3A4 to the N-monoethyl and N-desethyl products, confirming that the immediate vicinity of the heme iron is one binding site. In the presence of CYP3A4, steady-state fluorescence emission and excitation spectra, as well as excited-state lifetimes at varying Nile Red concentrations, indicate a high-affinity site that modulates the fluorescent properties of Nile Red. The Nile Red binding site is competitively eliminated by itraconazole, which is a high-affinity ligand known to coordinate to the heme iron. Together, the data suggest that Nile Red binds to the active site with high affinity ( approximately 50 nM), where it is desolvated in a low-dielectric environment. In addition, Nile Red is sequentially oxidized at rates comparable to or faster than those of other in vitro probes, which emphasizes its utility in the further examination of this important kinetic phenomenon in vitro.

Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements

Synchrotrons are now producing thousands of macromolecular structures each year. The need for complementary techniques available on site has progressively emerged, either to assess the relevance of the structure of a protein or to monitor changes that may occur during X-ray diffraction data collection. Microspectrophotometers in the UV–visible absorbance or fluorescence mode have evolved over the past few decades to become the instruments of choice to perform such tests. Described here are recent improvements to the microspectrophotometer of the so-called Cryobench laboratory located at the European Synchrotron Radiation Facility, Grenoble, France. Optical and mechanical properties have been enhanced so as to record better spectra on smaller samples. A device has been implemented to measure the signal decay of fluorescent samples, either in the crystalline or in the solution state. Recording of the fluorescence lifetime in addition to the steady-state fluorescence emission spectrum allows precise monitoring of the fluorescent sample under study. The device consists of an adaptation of a commercially available time-correlated single-photon-counting (TCSPC) system. A method to record and analyze series of TCSPC histograms,e.g.collected as a function of temperature, is described. To validate the instruments, fluorescence lifetimes of fluorescent small molecules or proteins in the crystalline or solution state, at room and cryo temperatures, have been measured. Lifetimes of a number of fluorescent proteins of the GFP family were generally found to be shorter in crystals than in solution, and slightly longer at cryo temperatures than at ambient temperature. The possibility of performing fluorescence lifetime measurements on crystals at synchrotron facilities widens the variety of spectroscopic techniques complementing X-ray diffraction on macromolecular crystallography beamlines.

Intramolecular Proton-Transfer Processes Starting at Higher Excited States: A Fluorescence Study on 2-Butylamino-6-methyl-4-nitropyridine N-Oxide in Nonpolar Solutions

This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M). It is known from the literature that this compound may undergo excited-state intra- or intermolecular proton-transfer reactions. In nonpolar solvents, 2B6M exhibits an unusual fluorescence behavior: there is a substantial difference between the relative band intensities of the excitation and absorption spectra. Furthermore, in emission two bands are observed, and their relative intensities depend on the excitation wavelength, thus violating the Kasha-Vavilov rule. It is the objective of this research to interpret these results. For this purpose, steady-state fluorescence excitation and emission spectra in the liquid state were recorded and quantum yields were determined for the two types of emission. In addition, absorption spectra were measured at room temperature and under low-temperature conditions. Finally, fluorescence lifetimes of the emitting species were determined using the time-correlated single photon counting technique. The results suggest that in the liquid state only one (monomeric) ground state species dominates, which can emit via two different pathways (from the normal and the tautomeric excited state). The excitation spectra point at two different internal proton-transfer processes, one starting at the S1 state and one starting at the S2 state. On the basis of the measured lifetimes and fluorescence quantum yields, a kinetic scheme was completed that can quantitatively explain the observations.

Changes in the energy distribution in mutant thylakoid membranes of pea with modified pigment content. II. Changes due to magnesium ions concentration

Low-temperature (77 K) steady-state chlorophyll fluorescence emission spectra, room temperature fluorescence and light scattering of thylakoid membranes isolated from pea mutants were studied as a function of Mg 2+ concentration. The mutants have modified pigment content and altered structural organization of the pigment-protein complexes, distinct surface electric properties and functions. The analysis of the 77 K emission spectra revealed that Mg 2+-depletion of the medium caused not only an increased energy flow toward photosystem I in all investigated membranes but also changes in the quenching of the fluorescence, most probably by internal conversion. The results indicated that the macroorganization of the photosynthetic apparatus of mutants at supramolecular level (distribution and segregation of two photosystems in thylakoid membranes) and at supermolecular level (stacking of photosystem II supercomplexes) required different Mg ion concentrations. The data confirmed that the segregation of photosystems and the stacking of thylakoid membranes are two distinct phenomena and elucidated some features of their mechanisms. The segregation is initiated by changes in the lateral microorganization of light harvesting complexes II, their migration (repulsion from photosystem I) and subsequent separation of the two photosystems. Most likely 3D aggregation and formation of macrodomains, containing only photosystem II antenna complexes, play a certain precursory role for the increasing degree of the membrane stacking and the energy coupling between the light harvesting complexes II and the core complexes of photosystem II in the frame of photosystem II supercomplexes.

Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membrane-bound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic alpha-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic alpha-helix (3.7 +/- 0.1 residues per turn and 97 +/- 3.0 degrees, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.

Carbon Nanotubes with Covalently Linked Porphyrin Antennae: Photoinduced Electron Transfer

Single- and multiwalled carbon nanotubes have been covalently functionalized with free-base porphyrin. The quantity of porphyrin linked to the surface was determined from thermogravimetric and UV-vis analysis. A reversible protonation equilibrium between the attached porphyrin and the residual acid groups of the carbon nanotubes has been identified. Steady-state fluorescence emission spectrum of the solutions of porphyrin-linked carbon nanotubes shows that the porphyrins act as energy absorbing and electron transferring antennae, and the carbon nanotubes act as efficient electron acceptors. The porphyrin-linked carbon nanotubes show 95-100% emission quenching, indicating a fast photoinduced electron transfer.

Potential of Albumin Nanoparticles as Carriers for Interferon Gamma

Although interferon gamma (IFN-γ has been extensively studied as a potent activator for macrophages and as a promising adjuvant in vaccines, its rapid biodegradation and clearance have severely limited its clinical efficacy. Our major objective in this work was to develop formulation conditions to get high association of the cytokine to albumin nanoparticles, without leading any conformational changes and subsequent loss of activity. To achieve this objective, two different formulations were prepared by either 1) incubation between the cytokine and the newly prepared nanoparticles (IFN-NPA) or 2) between the protein and IFN-γ prior coacervation (IFN-NPB). Steady-state fluorescence emission spectra revealed that the environment of the tryptophan (Trp) residue was not affected by conditions of mechanical stress required for preparing nanoparticles. A bioassay for antiproliferative activity with Hela cells indicated that the cytokine, after their desorption from the surface of nanoparticles (IFN-NPA), fully retained its activity. It also indicated that the cytokine was principally associated with nanoparticles via electrostatic interactions and confirmed by desorption experiments carried out in media with different pH and ionic strength, with burst effect ranked in the order pH 5 > pH 7.4 > pH 8.5. Also, the adsorption of IFN-γ onto these carriers was able to improve the priming effects of IFN-γ on the nitric oxide production (NO) by RAW macrophages. On the contrary, when we incubated the cytokine with the albumin solution prior to the desolvation process for preparing nanoparticles (IFN-NPB), we obtained better encapsulation efficiencies (around 100%), but the cytokine was inactive: it was not detected by ELISA or bioassay in Hela cells and unable to stimulate NO production by macrophages.

The Isoindazole Nucleus as a Donor in Fullerene‐Based Dyads. Evidence for Electron Transfer.

A series of isoindazole-C60 dyads 4a−c based on pyrazolino[60]fullerene have been prepared by 1,3-dipolar cycloadditions of the nitrile imines, generated in situ from hydrazones 3a−c, to C60. Molecular orbital calculations for 4b revealed that the electron distribution of the HOMO is located on the isoindazole moiety, while the electron distribution of the LUMO is located on the C60 moiety. Electrochemical properties of the new dyads 4a−c show a similar electron affinity with respect to C60. Charge-transfer interactions in the ground state between the isoindazole ring and the fullerene cage are predicted by the molecular orbital calculations and confirmed by electrochemical studies in 4a,b. Steady-state fluorescence emission spectra of dyads 4a−c show that fluorescence intensities in polar benzonitrile solvent decrease with increasing electron-donating ability of the substituent attached on the isoindazole group. This was confirmed by the shortening of fluorescence lifetimes, from which intramolecular cha...

Temperature and cholesterol composition-dependent behavior of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]- sn-glycero-3-phosphocholine in 1,2-dimyristoyl- sn-glycero-3-phosphocholine membranes

We present a steady-state and time-resolved fluorescence emission spectra analysis of the membrane probe 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]- sn-glycero-3-phosphocholine (DANSYL) in 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and cholesterol multi-lamellar vesicles (MLV) prepared by modified rapid solvent exchange. We report that the dose-dependent cholesterol-induced blue shifts in the steady-state fluorescence emission spectra observed in DMPC MLV are due to complex solvent effects that include time-dependent dipolar relaxation and the formation of internal charge transfer (ICT) states. A key finding of this investigation is identification of two distinguishable DANSYL populations existing at both shallow and deep locations in the membrane; these two DANSYL populations are evidence of laterally phase-separated domains at cholesterol compositions between X chol = 0.30 and 0.60 at 30 °C in DMPC MLV.

The isoindazole nucleus as a donor in fullerene-based dyads. Evidence for electron transfer.

A series of isoindazole-C(60) dyads 4a-c based on pyrazolino[60]fullerene have been prepared by 1,3-dipolar cycloadditions of the nitrile imines, generated in situ from hydrazones 3a-c, to C(60). Molecular orbital calculations for 4b revealed that the electron distribution of the HOMO is located on the isoindazole moiety, while the electron distribution of the LUMO is located on the C(60) moiety. Electrochemical properties of the new dyads 4a-c show a similar electron affinity with respect to C(60). Charge-transfer interactions in the ground state between the isoindazole ring and the fullerene cage are predicted by the molecular orbital calculations and confirmed by electrochemical studies in 4a,b. Steady-state fluorescence emission spectra of dyads 4a-c show that fluorescence intensities in polar benzonitrile solvent decrease with increasing electron-donating ability of the substituent attached on the isoindazole group. This was confirmed by the shortening of fluorescence lifetimes, from which intramolecular charge-separation rates and efficiencies via the excited singlet states of the fullerene moiety were evaluated. The yields of the triplet states in polar solvent decrease with the electron-donating ability, supporting the competitive formation of the charge-separated state with the intersystem crossing from the excited states. Thus, isoindazole[60]fullerene 4b can be considered a molecular switch with an AND logic gate.

Effects of Subzero Temperatures on Fluorescent Probes Sequestered within Aerosol-OT Reverse Micelles

We report on the effects of temperature (+30 to −100 °C) and water loading on the steady-state fluorescence from four fluorescent probe molecules when they are sequestered within the interior of sodium bis(2-ethyl-1-hexyl) sulfosuccinate (Aerosol OT, AOT) reverse micelles. Each probe locates itself to different average microenvironments within the reverse micelle, and these individual microenvironments behave differently to changes in the molar ratio of water-to-AOT (R) and temperature. Changes in probe steady-state fluorescence emission spectra or fluorescence anisotropy indicate that “freezing” occurs within the water pool at temperatures between −10 and −60 °C. The lowest freezing points are generally observed at the lowest R values.

A Fluorescent Probe-labeled Escherichia coli Aspartate Transcarbamoylase That Monitors the Allosteric Conformational State

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.