Fluorescence spectroscopy kinetic measurements

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

A rapid reaction kinetic technique (time scale = 10-1000 ps) that typically uses a Van de Graff accelerator or a microwave linear electron accelerator to promptly generate a pulse of electrons at sufficient power levels for excitation and ionization of target substances by electron impact. The technique is the direct radiation chemical analog of flash photolysis and the ensuing kinetic measurements are accomplished optically by IR/visible/UV adsorption spectroscopy or by fluorescence spectroscopy. 

In this work we utilized FTIR methods to examine the SA monolayers on flat, polar solid surfaces prepared from nonpolar solutions. We used ATR and GI FTIR measurements to characterize the material and bonding of the S A monolayers, and used transmission and ATR FTIR to monitor the dynamics of the SA adsorption process. With reference to measurements on standard Langmuir-Blodgett monolayer samples, we were able to quantify the S A kinetic results. We also used fluorescence spectroscopy of incorporated pyrene probes in S A mixed monolayer films as a simple method for the determination of the relative adsorption and thermodynamic constants. 

Fluorescence Correlation Spectroscopy and Fluorescence Burst Analysis. Several nanoscopic chemical imaging approaches work very well for measurements of chemical kinetics, interactions, and mobility in solution. Fluorescence correlation spectroscopy (FCS) measures the temporal fluctuations of fluorescent markers as molecules diffuse or flow in solution through a femtoliter focal volume.54 Their average diffusive dwell times reveal their diffusion coefficients, and additional faster fluctuations can reveal chemical reactions and their kinetics if the reaction provides fluorescence modulation. Cross-correlation of the fluorescence of two distinguishable fluorophore types can very effectively reveal chemical binding kinetics and equilibria at nanomolar concentrations. 

Time-resolved fluorescence spectroscopy and fluorescence anisotropy measurements have been applied to study (i) excimer formation and energy transfer in solutions of poly(acenaphthalene) (PACE) and poly(2-naphthyl methacrylate) (P2NMA) and (ii) the conformational dynamics of poly(methacrylic acid) (PMA) and poly (acrylic acid) as a function of solution pH. For PACE and P2NMA, analysis of projections in which the spectral, temporal and intensity information are simultaneously displayed have been used to re-examine kinetic models proposed to account for the complex fluorescence decay behaviour that is observed. Time-resolved fluorescence anisotropy measuranents of fluorescent probes incorporated in PMA have led to the proposal of a "connected cluster" model for the hypercoiled conformation of this polymer existing at low pH. 

Fluorescence correlation spectroscopy (FCS) was used to elucidate the association kinetics of lacZ fragments derived from E. coli (3-galactosidase. This example demonstrates the power of FCS to provide mechanistic information by allowing the interaction to be observed at the molecular level. In general, it demonstrates how FCS may be used as a powerful tool for kinetic measurements in the life sciences. 

X-ray fluorescence spectroscopy has been used to determine 50 ppb of nickel and vanadium after they have been concentrated on ion exchange resins (5, 6). Emission spectroscopy has been used but is only semi-quantitative at the nanogram/gram levels of interest to the Project. Nevertheless, the technique may be useful as a screening tool. Two relatively new instrumental techniques—spark source mass spectrometry (7) and kinetics of metal-catalyzed reactions (8)—can measure extremely low levels of nickel and vanadium, but they have not been utilized to any appreciable extent. 

It has been known for over 40 years that the renaturation of short guanine rich oligomers such as G3 is very slow. Absorbance and fluorescence spectroscopy were used to measure kinetics of association. In agreement with the seminal paper by Wyatt et who used size exclusion chromatography to study... 

The possible SLI variables for fluorescence spectroscopy are (1) excitation wavelength, (2) emission wavelength, (3) some environmental variable which alters relative concentration, and (4) one of the following (a) some environmental variable (such as quencher concentration) which alters the fluorescence quantum yield, (b) time since pulse excitation, or (c) modulation frequency. Quencher concentration and time since excitation cannot be separate SLI variables because a change in fluorescence quantum yield affects decay kinetics. However, it may be profitable to make measurements of decay kinetics at several different quencher concentrations and then combine time and quencher concentration to form a single SLI variable. 

An acetyl and an amide group block the N and C termini of the peptide chain. The tryptophan residue is added as a probe to collect time-resolved fluorescence signal under nanosecond T-jump spectroscopy, allowing measurement of coil to helix transition. The experimentally estimated relaxation time at room temperature for this transition is about 300 ns. The inverse of the experimental relaxation time is the sum of two rate constants, from the unfolded to the folded state and back. The equilibrium constant of this transition is about 1, which indicates that the forward and the backward rates are almost the same. The experimental first passage time from the folded to the unfolded state (which we estimate computationally in this chapter) is therefore 600 ns. This timescale seems achievable within the standard model and atomically detailed simulations. However, one should keep in mind that an ensemble of trajectories is required to study kinetics. The calculation of kinetics will be at least 100 times more expensive than the calculation of a single trajectory and therefore difficult to do with the usual standard model. 

Besides fluorescence spectroscopy, time-resolved spectroscopy can rely on the measurement of excited (singlet or triplet) state absorption. Similarly to ground-state absorption, the spectral and absorbance properties may be altered by CyD complexation and yield information about the behavior of the complex in the excited state in addition, the time dependence (formation and decay) of the excited state absorption yields information about the kinetics and dynamics of the system. This is illustrated by the behavior of the lowest triplet state of naphthalene as measured by nanosecond spectroscopy using a Q-switched Nd YAG laser at 266 nm for excitation [21]. The triplet-triplet absorption spectra were measured in neat solvents (water and ethanol) and in the presence of a- and -CyD (Fig. 10.3.3). The spectra in ethanol and H2O had the same absorption maximum, but the transition was considerably weaker and broadened in H2O. Both CyDs induced a red shift, and a-CyD additionally narrowed the main band considerably. Fig. 10.3.4 shows the effect of a-CD concentration on the time evolution of the triplet-triplet absorption at 416 nm in the microsecond range. Triplet decay was caused by O2 quenching a detailed kinetic analysis of the time dependence yielded two main components which could be assigned to the free guest and the 1 2 complex, in full... 

The high sensitivity of fluorescence spectroscopy and the selectivity of enzymatic assays are responsible for the increasing use of fluorimetric methods in enzymology. Enzyme determinations usually involve the use of kinetic methodology for measuring the rate of formation of the fluorescent product, while both equilibrium and kinetic methods are used to determine the substrates. Fluorimetric measurements on enzyme-catalyzed reactions have been used for a long time to determine a variety of enzymes and substrates (Figure 2). 

A general discussion of the use of least-squares fitting in fluorescence measurements may be found in (28). The global analysis of fluorescence data is discussed in (29). Commercially available time-resolved fluorimeters are typically sold with data analysis software included. Available stand-alone packages include the Globals Unlimited suite, which is capable of analysing both time- and frequency-domain data, stopped-flow kinetics, etc. The Center for Fluorescence Spectroscopy at the University of Maryland (USA) also offers software for frequency- and time-domain fluorescence lifetime analysis. 

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