Temperature jump techniques example

To circumvent the equilibrium requirement, which is the greatest limitation of temperature jump, attempts have been made to combine this technique with some others. The stopped-flow temperature jump, for example, has found use in studies of reactions involving the formation of intermediates on not-too-short timescales (>10 ms) [23]. In this method, the temperature jump is applied during the course of the stopped-flow reaction. The equilibrium between the reactants and intermediates is perturbed, which permits a direct study of the fast steps occurring prior to the rate-determining step [28]. 

To study rates of antibody-hapten reactions the principal methods employed have involved stopped flow or temperature jump techniques. The former was first used by Sturtevant et al. (64) and Day et al. (65). The temperature jump method has been employed by Froese, Sehon, and their collaborators (66-68) and more recently by Pecht et al. (69). Both methods are utilized in conjunction with very rapid optical measurements (in the millisecond range). For example, Sturtevant et al. took advantage of a spectral shift which occurs upon combination of anti-Dnp antibody with the dye, 2-(Dnp-azo)-I-naphthol-3,6-disulfonic acid (64). With the same hapten, and with e-Dnp-L-lysine and e-Dnp-6-aminocaproate. Day et al. (65) used the method of fluorescence quenching (Section VI,D) with a stopped flow apparatus. In the temperature jump technique the components are first equilibrated, a temperature increment is rapidly induced (up to 10°C in 0.1 isecond), and the rate of reequilibration at the new temperature is measured. Velocity constants can be estimated from the data the mathematical approaches required are described in the references cited. 

The dynamics of intercalation of small molecules with DNA, groove binding and binding to specific sites, such as base pair mismatches have been studied by stopped-flow,23,80 108 temperature jump experiments,26,27,94 109 120 surface plasmon resonance,121 129 NMR,86,130 135 flash photolysis,136 138 and fluorescence correlation spectroscopy.64 The application of the various techniques to study the binding dynamics of small molecules will be analyzed for specific examples of each type of binding. 

Studies on the dynamics of complexation for guests with cyclodextrins have been carried out using ultrasonic relaxation,40 151 168 temperature jump experiments,57 169 183 stopped-flow,170,178,184 197 flash photolysis,57 198 202 NMR,203 205 fluorescence correlation spectroscopy,65 phosphorescence measurements,56,206 and fluorescence methods.45,207 In contrast to the studies with DNA described above, there are only a few examples in which different techniques were employed to study the binding dynamics of the same guest with CDs. This probably reflects that the choice of technique was based on the properties of the guests. The examples below are grouped either by a type of guest or under the description of a technique. 

Monitoring of events following perturbations can be achieved in much shorter times by photolysis. A variety of monitoring techniques have been linked to both methods (Table 3.7). It is valuable to obtain kinetic data by more than one method, when possible. The measurement of spin-change rates have, for example, been carried out by a variety of rapid-reaction techniques, including temperature-jump, ultrasonics and laser photolysis with consistent results (Sec. 7.3). 

The kinetics and dynamics of crvptate formation (75-80) have been studied by various relaxation techniques (70-75) (for example, using temperature-jump and ultrasonic methods) and stopped-flow spectrophotometry (82), as well as by variable-temperature multinuclear NMR methods (59, 61, 62). The dynamics of cryptate formation are best interpreted in terms of a simple complexation-decomplexation exchange mechanism, and some representative data have been listed in Table III (16). The high stability of cryptate complexes (see Section III,D) may be directly related to their slow rates of decomplexation. Indeed the stability sequence of cryptates follows the trend in rates of decomplexation, and the enhanced stability of the dipositive cryptates may be related to their slowness of decomplexation when compared to the alkali metal complexes (80). The rate of decomplexation of Li" from [2.2.1] in pyridine was found to be 104 times faster than from [2.1.1], because of the looser fit of Li in [2.2.1] and the greater flexibility of this cryptand (81). At low pH, cation dissociation apparently... 

Transient technique — A technique whose response is time dependent and whose time dependence is of primary interest, e.g., -> chronoamperometry, -> cyclic voltammetry (where current is the transient), -> chronopotentiometry and -> coulostatic techniques (where voltage is the transient). A transient technique contrasts with steady-state techniques where the response is time independent [i]. Some good examples are cyclic voltammetry [i, ii] (fast scan cyclic voltammetry), the indirect-laser-induced-temperature-jump (ILIT) method [iii], coulostatics [i]. The faster the transient technique, the more susceptible it is to distortion by -> adsorption of the redox moiety. 

Let me give you another example. What happens when a laser beam is striking a molecular surface I have found out that the laser can heat the surface at a very rapid rate of about 10 -10 degrees per second. Of course, this does not last long otherwise, we would have a thermonuclear reaction But even for a millionth of a second, the temperature jump is huge, and the molecules hop off the surface into the gas phase, often without fragmenting. Then we come in with a second laser and we ionize those molecules that absorb a particular color of that second laser. Because we now have ions, we can do time-of-flight mass spectrometry. Laser-desorption/laser-ionization mass spectrometry is an invention of ours, and many other people have worked on this technique too. 

The presteady-state region requires knowledge of the forward (kf) and backward (k, ) rates in the formation and reversal of enzyme-substrate complexation. Most commonly such studies involve fast-mixing techniques (e.g. stopped-flow). Of course, for very fast kf and k, processes, the ratio of kf and kf is effectively an equilibrium constant. This simplification allows the use of special relaxation techniques (e.g. temperature jump) and gives access to some very rapid steps which could not otherwise be probed. In addition, the number of component rate processes in kf or kb can be assessed using such procedures. There are several examples of enzymes which use a string of enzyme substrate complexes, as depicted in Eqn. 5. 

At the suggestion of one reviewer, we oflFer a brief discussion of the techniques used in proposing a mechanism for a given reaction from kinetic data obtained by rapid reaction techniques. We use the case of the reaction of fluoride with HRP as an example. The proton reactions are too rapid to observe on the time scale of a conventional temperature-jump apparatus (32). These proton reactions would couple the three paths for fluoride binding and dissociation proposed in Mechanism I so that only one relaxation time, t, is observed. At any given pH, the relation between t and the constants in the above mechanism is... 

This is the simplest mechanism leading to Michaelis-Menten kinetics, but many other mechanisms also do so. To investigate mechanisms in more detail it is necessary to use special techniques for studying very rapid reactions, such as the stopped-flow and temperature-jump methods (Section I). Various factors give deviations from Michaelis-Menten kinetics. For example, sometimes the complex ES adds on an additional substrate molecule to give ES2 if this does not react as rapidly as ES there is a falling off of the rate at higher substrate concentrations. 

Whenever a chemical equilibrium is subjected to a perturbation, most commonly a change in temperature, pressure, pH, or other concentrations, the system will start to relax back to a new equilibrium state. The kinetics of this relaxation can be followed. Methods for quickly inducing a perturbation followed by monitoring the relaxation are referred to as jump techniques. Changes in temperature, pH, and pressure can often be done fast enough that reactions with half-lives in the microsecond range can be followed. For example, the equilibrium positions of Bransted acid-base reactions are controlled by the pH, and therefore pH jump experiments are particularly useful with these reactions. 

Kinetic studies of micelle formation and dissociation by direct methods are scarce as already mentioned by Tuzar and co-workers [7,114] and later on by Hamley [11]. Informations can be obtained by fast reaction techniques, such as stop-flow, temperature or pressure jump techniques, as well as by steady state methods, for example ultrasonic absorption, NMR, ESR. 

As emphasized in the introduction to relaxation kinetics, the methods described in this section can, in principle, be extended to derive equations for mechanisms with any number of relaxation times. Qearly these become progressively more complex as the number of roots increases. Assumptions have to be made in terms of limiting conditions, to extract useful information from them. The practical difficulties of resolving multiple exponentials from noisy experimental records have been alluded to before and helpful hints on this topic are presented in section 2.3. The discussion of examples of investigations by temperature and pressure jump techniques in... 

Of particular interest is the use of uv-visible spectrophotometry, since there is the added possibility of following the kinetics of simple hydrogen-bond formation by means of the temperature-jump relaxation technique with a conventional optical detection system. The method is suitable for acid or base species which contain a suitable chromophore, for example 2,4-dinitrophenol (2,4-DNP),... 

---