Burst phase

Stopped-flow CD studies of protein folding have generally revealed a species that forms within the dead-time of the experiment, 10 ms (Kuwajima et al., 1985, 1987). The 222 nm ellipticity of this so-called burst-phase intermediate is generally substantially more negative than... 

The burst-phase intermediate has been studied intensively in the folding of bovine o -lactalbumin (Arai and Kuwajima, 1996 Ikeguchi et al., 1986 Kuwajima et al., 1985). The CD spectrum of the intermediate has been determined by measuring the folding kinetics at various wavelengths and extrapolating the CD to zero time. The results are shown as open circles (holoprotein) and squares (apoprotein) in Figure 35. The... 

Relative Amplitude of Burst Phase Measured by Time-Resolved Far-UV CD Spectroscopy for Various Globular Proteins a... 

The burst phase amplitude represents the percentage change in the far-UV CD signal (216-225 nm) occurring in the dead time of the stopped-flow experiment relative to the total change on folding. 

Qi et al. (1998) have demonstrated that ribonuclease A exhibits behavior like that of cytochrome c. The burst phase observed on dilution of Gdm HCl-denatured RNase A is mimicked exactly by reduced RNase A. The latter, when carboxamidomethylated to prevent oxidation, has a CD at 222 nm that is nearly independent of temperature and indicative of extensive unfolding at zero denaturant. 

Fig. 42. The unfolded baseline and the Cyt c burst phase. The solid curves show the equilibrium behavior of Cyt c. The equilibrium fluorescence and CD of the (unfolded) fragments (A and <>) match the unfolded holo Cyt c baseline at high GdmCl and define the continuation of the unfolded baseline to lower GdmCl concentrations. The horizontal dashed line shows the initial fluorescence and CD in the stopped-flow experiments (4.3 M GdmCl). The solid symbols indicate the fluorescence (A) and the ellipticity at 222 nm (B) reached by holo Cyt c in the burst phase on dilution into lower (or higher) GdmCl, as suggested by the arrows (starting from either pH 2 ( ) or pH 4.9 ( )). These comparisons are made on an absolute, per-molecule basis. Forster-averaged distance (Trp-59 to heme) is at the right of A. (From Sosnick et al., 1997, with permission. 1997, National Academy of Sciences, USA.)...

For amide substrates, however, no burst phase can be observed. It must, therefore, be concluded that the rate-limiting step is not the same for amides... 

A device consisting of an array of frustum-shaped cells that contain a drug dispersed in a permeable matrix is shown to obey zero-order release kinetics following an initial burst phase. Geometric shapes of dissolving solids or diffusion systems and the constraints of impermeable barriers influence mass transport and can be exploited as in the constant release wedge- or hemispheric-shaped devices. 

The results of experimental release studies from devices loaded with 0.25%, 1%, and 4% suspension concentrations are shown in Figure 10. The corresponding simulated profiles are given on Figure 11. There is a reasonable correlation between the experimental and theoretical results regarding the effect of suspension concentration on the amount released and on the burst phase. 

Summary. A device comprised of an array of frustum-shaped cells in which drug was dispersed provided a release pattern having a burst phase followed by a constant release rate. This pattern results from the geometric shape of the cells. A mathematical model was developed to predict the release characteristics of this system. 

Fig. 1.2. Correlation between proton occupancies in the kinetic burst phase intermediate (black circles) and average area buried upon folding (AABUF, gray lines) for wild-type apomyoglobin and for a quadruple mutant (LeullGly, Trpl4Gly, Ala71Leu, Gly73Trp - termed the GGLW mutant). Reproduced with permission from [10]...

If turnover is measured with a very high concentration of substrate relative to that of enzyme (in practice, this means at very low enzyme concentrations) then after a short pre-steady state (or burst) phase the rate of turnover is constant. In this steady state region, the concentration of enzyme-substrate complex is constant and the rate of reaction is given by the following equation ... 

The mere observation of a burst implies that a step after chemistry is at least partially rate limiting for steady-state turnover. In addition, if no observable burst occurs, then the data imply that chemistry is largely rate limiting. When a burst can be observed, quantitative fitting of the amplitude and rate of the burst phase relative to the steady-state phase affords estimates for three rate constants k2, k-2 and ks in the pathway shown above (Scheme 2). 

Conceptual Insights, Enzyme Kinetics. See the section entitled "Pre-Steady-State Kinetics" in Conceptual Insights module to better understand why a "burst" phase at short reaction times implies the existence of an enzyme-substrate intermediate. 

Alternatively, all four ATPs may bind, accumulating subtle conformational changes in the ring which trigger hydrolysis only once all of the ATPs are bound. Moreover, it is also possible that the ATPs are hydrolyzed in the burst phase, immediately before each subunit generates a step. Finally, it is also possible that the hydrolysis of ATP is decoupled between the subunits and that it can occur spontaneously on its own, independent of the chemical state of the rest of the ring. The burst structure would then be maintained by another kinetic state, a bottleneck state, which would force the motor to wait until the necessary hydrolysis had occurred. A similar set of arguments can be made for the location of ADP release, with the result that ADP may be released in either the burst or dwell phase. 

Fig. 3.5 Wrapping patterns for chain conformations occurring during the burst phase (cf. Fig. 3.4) of protein-G variant generated at 6.4 x 10 4 s upperpanel) and 6.5 x 10 4 s lowerpanel). These conformations commit the chain to fold are part of the transition state ensemble, and do not contain exclusively native interactions, as it becomes apparent in the upper panel. Reprinted from [35], with permission from Elsevier...

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