Reaction velocities measurement

If the inhibition is found to be rapidly reversible, we must next determine if the approach to equilibrium for the enzyme-inhibitor complex is also rapid. As described in Chapter 4, some inhibitors bind slowly to their target enzymes, on a time scale that is long in comparision to the time scale of the reaction velocity measurement. The effect of such slow binding inhibition is to convert the linear progress curve seen in the absence of inhibitor to a curvilinear function (Figure 5.10). When nonlinear progress curves are observed in the presence of inhibitor, the analysis of... 

These considerations indicate that the thermodynamic analysis of the Zr system must not be restricted only to the processing temperature of 1900°C. The kinetics of this reaction have been studied using thermo-gravimetic analysis (TGA), interrupted growth experiments, ART calculations, and reaction velocity measurements. The proposed reaction mechanism is as follows [36] ... 

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

The turnover number of an enzyme, is a measure of its maximal catalytic activity, is defined as the number of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is saturated with substrate. The turnover number is also referred to as the molecular activity of the enzyme. For the simple Michaelis-Menten reaction (14.9) under conditions of initial velocity measurements, Provided the concentration of... 

ASSAYS OF ENZYME-CATALYZED REACTIONS TYPICALLY MEASURE THE INITIAL VELOCITY... 

The method requires less time since the reaction velocity is measured directly. 

It has been well known for a long time that some reactions taking place at a very slow rate may be markedly accelerated by the simultaneous occurrence of another reaction of measurable velocity. On the suggestion of Kessler this phenomenon is called chemical induction and it is said that the reaction of measurable velocity induces the other slow reaction. 

To determine the value of y, Yonetani and Theorell suggest measuring reaction velocity at several fixed concentrations of one inhibitor while titrating the second inhibitor. The reciprocal of velocity (l/v0) is then plotted as a function of concentration for the titrated inhibitor (Figure 3.11). If the two compounds are binding in a mutually exclusive fashion, this type of plot results in a series of parallel lines (Figure 3.11A). If the two compounds bind independently (y = 1) the lines in the... 

The initial velocity of reaction is defined by the slope of a linear plot of product (or substrate) concentration as a function of time (Chapter 2), and we have just discussed the importance of measuring enzymatic activity during this initial velocity phase of the reaction. The best measure of initial velocity is thus obtained by continuous measurement of product formation or substrate disappearance with time over a convenient portion of the intial velocity phase. However, continuous monitoring of assay signal is not always practical. Copeland (2000) has described three types of assay readouts for measuring reaction velocity continuous assays, discontinuous... 

Again, if we wish to measure the effects of an inhibitor on enzyme activity, we must cast Equation (A2.16) in terms of reaction velocity. Combining Equation (A2.13) with Equation (A2.16), we obtain... 

The energetics of the Eley-Rideal reaction (A E —230 kJ mol-1) are well established.42 Here, the highly exoergic reaction forming gas-phase HC1 was probed by time-of-flight velocity measurements,39,41 scattering angular distributions,39,41 and state-selective laser spectroscopy.39-41... 

NMR measurements of reaction velocities and equilibrium constants as a function of temperature. 3, 187... 

Reaction mechanisms, in solution, entropies of activation and, 1, 1 Reaction mechanisms, use of volumes of activation for determining, 2,93 Reaction velocities and equilibrium constants, NMR measurements of, as a function of temperature, 3, 187... 

The velocity is not necessarily the same at all times after you start the reaction. The depletion of substrate, inhibition by the product, or instability of the enzyme can cause the velocity to change with time. The initial velocity is measured early, before the velocity changes. Initial velocity measurements also let you assume that the amount of substrate has not changed and is equal to the amount of substrate that was added. 

The reaction velocity is dependent on temperature, which must therefore be measured. According to Nyman (N5) and judging from our own experience, the method is sensitive down to a HbBC of 5 mg/100 ml. 

It is therefore a fundamental principle in the estimation of every enzyme that the rates measured should be maximal initial reaction velocities, tangents to progress curves at zero time. They really should represent the amount of enzyme protein in the reaction mixture. 

The measurement of reaction velocity is most frequently done in one of three ways. The velocity of the reaction in terms of substrate depletion or... 

---