PH-jump method

Fig. 23 A plot of the observed pseudo-first-order rate constant for the methanolysis of 0.04mM HPNPP ( , left axis) catalyzed by 0.2mM35 2Zn(II) or 0.04mM methyl /j-nitro-phenyl phosphate (O, right axis) catalyzed by 0.4 mM 35 Zn(II) as a function of the [CH30-]/ [35 Zn(II)] ratio at 25 + 0.1 °C. Experiments done by pH jump method starting at a [CH30-]/ [35 Zn(II)] ratio of 1.0 (vertical dashed line, (pH = 9.5) and adding acid (left) or base (right). Reproduced with permission from ref. 95.

The pKj values obtained for PCu(II) and PCu(I) are summarized in Table 9. Before discussing what appears to be a clear cut trend, the precision of individual pKa s should first be considered. It has for example been necessary to revise (downwards) the spinach PCu(II) pKa from 5.3 to 4.8 [121]. Plastocyanin is known to denature at or around pH 4.0 and in earlier work the lowest pH used was 4.5, which does not allow as accurate a fit to pK, values. By using a pH-jump method in which the final low pH is attained at the time of stopped-flow mixing (one reactant solution carrying substantially more buffer is allowed to control the pH), it is now possible with some confidence to include data down to pH 4.0. The other uncertainty is in the precision of the pKg for the PCu(I) remote site from a two pK fit. Again the data has to be free from artifacts introduced at... 

The dynamics of proton binding to the extra cellular and the cytoplasmic surfaces of the purple membranes were measured by the pH jump methods [125], The purple membranes selectively labeled by fluorescein Lys-129 of bacteri-orhodopsin were pulsed by protons released in the aqueous bulk from excited pyranine and the reaction of the protons with the indicators was measured. Kinetic analysis of the data implied that the two faces of the membrane differ in then-buffer capacities and in their rates of interaction with bulk protons. The extracellular surfaces of the purple membrane contains one anionic proton binding site per protein molecule with pA" 5.1. This site is within a Coulomb cage radius from Lys-129. The cytoplasmic surface of the purple membrane bears four to five pro-tonable moieties that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3 X 1010 M-1 sec ). The proximity between the elements is sufficiently high that even in 100 mM NaCl, they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. Quantitative evaluation of the dynamics of proton transfer from photoactivated bacteriorhodopsin to the bulk has been done by using numerical... 

With [Co(phen)3] + as oxidant for PCu(I), rate constants determined by the stopped-flow method approach zero at low pH, consistent with zero reactivity of the trigonally coordinated Cu(I) form. However, with [FelCNlel as oxidant there is sometimes difficulty in fitting rate constants to the relevant [H ] dependence, which leaves open the question as to whether the rates actually become zero (57). Whereas [Co(phen)3l + is believed to react with PCu(I) at both the remote (acidic) and the adjacent (hydrophobic) patches, [FefCNleP reacts predominantly at the latter. The instability of PCu(I) in solution at pH < 4.5 makes it difficult to settle this issue conclusively. The range of studies has been extended using the pH-jump method in which protein, at high pH (with relatively small concentration of buffer), is stopped-flow mixed with the redox reagent at low pH (with excess buffer), and this approach has been used more extensively in recent studies. 

Pines, E. Huppeet, D., Kinetics of proton transfer in ice via the pH-jump method evaluation of the proton diffusion rate in polycrystalline doped ice, Chem. Phys. Lett., 1985, 116, 295-301. 

The reversible unfolding of bovine a-lactalbumin in the presence of low concentrations of guanidinium hydrochloride has been examined by spectroscopic and pH-jump methods." Unfolding was shown to occur between the native and denatured states, since the apparent rate constants for unfolding and refolding depend only on pH. 

Concentration-jump methods, such as the pH-jump technique cited earlier, can be used in relaxation kinetics, but this approach is described later (Section 4.4). 

A number of methods have been used for determining Kg values cation selective electrodes, pH-metric methods, conductimetry, calorimetry, temperature-jump relaxation measurements, membrane conductance measurements, nuclear magnetic resonance, optical rotatory dispersion. The results listed in Tables 7—10 have been obtained by various methods and at different ionic strengths so they may not always be strictly comparable. However, the corrections are probably small and the experimental accuracy is generally the same or very similar within a certain ligand type. 

There are also nonthematic methods that allow the formation of acylenzymes under conditions where they are stable, so that they can be stored in a syringe in a stopped-flow spectrophotometer. For example, it is possible to synthesize certain nonspecific acylenzymes and store them at low pH.9 12 When they are restored to high pH, they are found to deacylate at the rate expected from the steady state kinetics. This approach has been extended to cover specific acylenzymes. When acyl-L-tryptophan derivatives are incubated with chymotrypsin at pH 3 to 4, the acylenzyme accumulates. The solution may then be pH-jumped by mixing it with a concentrated high-pH buffer in the stopped-flow spectrophotometer.1314 The deacylation rate has been measured by the proflavin displacement method and by using furylacrylolyl compounds. 

In many cases, the crystal retains enzymatic activity. In some cases, the activity of the enzyme in the crystal is the same as that in solution. The methods used for initiating reactions for study by the Laue method are used to measure activity. For example, pH-jump the acylenzyme indolylacryloyl-chymotrypsin was crystallized at a pH at which it is stable. On changing the pH to increase the reactivity, the intermediate was found to hydrolyze with the same first-order rate constant as occurs in solution the reactions of crystalline ras p21 protein, glycogen phosphorylase, and chymotrypsin have been initiated by photolysis.52 Glyceraldehyde 3-phosphate dehydrogenase has also identical reaction rates in the crystal and solution under some conditions.53... 

A protease-free assay using Suc-Ala-Ala-Pro-Phe-DFA (DFA is 2,4-difluoroani-line) was also developed, but its sensitivity at 246 nm is low (<0.006 absorbance unity) for a typical peptide concentration of 21 pmol L-1 [56], The same problem limits the applicability of the assay reported by Fischer and coworkers based on the pH sensitivity of the secondary amide bonds. A pH jump (i.e. from 2.0 to 7.3) led to a slight variation of absorbance at 220 nm which reflects the pH-dependent CTI (Fig. 8.8). However, this assay cannot be used routinely with oligopeptides, although it gives kinetic data that cannot be easily obtained by other methods [21]. 

The dependence of equilibrium constants for various hydrolysis and transfer reactions of ATP has been calculated as a function of pH, pMg, and temperature " . Temperature-jump methods have shown that substitution rates for metal binding to ATP are limited by the rate of dissociation of the first water to depart from the metal ion in an Swl-type reaction ". 

Gutman, M., The pH jump probing of macromolecules and solutions by a laser-induced, ultrashort proton pulse-theory and applications in biochemistry. Methods Biochem. Anal., 1984, 30, 1-103. 

Deprotonation kinetics of intramolecularly H-bonded dicarboxylic acid mono-anions are usually a good deal easier to study systematically by the temperature jump method because of their smaller pK values that do not require such high sample solution pH s as do the azo dyes of Table I. In Table II we have representative kinetic results for several dicarboxylic acids. The first nine acids in Table II are derivatives of malonic acid, HOOCCHg-COOH, substituted at the number two carbon. Their specific rates, for deprotonation by OH have been reported previously. The kinetic data... 

For example, the kinetics of Fe dissociation from Fe +nFbp(X) (nFbp = recombinant ferric binding protein from Neisseria meningitides, X = P04, citrate) driven by H+ were studied by pH-Jump stopped-flow methods. Researchers used this technique to induce Fe + dissociation from Fe + nFbp(X) (X = P04, citrate), which enabled researchers to study the mechanism in the absence of... 

In the absence of substrates, a relaxation process is observed in solutions of ribonuclease with the temperature jump method having a relaxation time of 0.1 to 1 msec. This relaxation time is independent of the enzyme concentration but is pH dependent. It can be attributed to an isomerization or conformational change of the enzyme, and a simple mechanism consistent with the data is... 

The important conclusions from this figure are (i) the pH at the equivalence point decreases with decreasing concentration, (ii) the pH jump around the equivalence point, defined as ApH between t = 0.99 and t = 1.01, decreases with decreasing concentration. The latter also means that the slope decreases with concentration, and thus the random error will increase, since the random error is always related to a constant ApH typical for a certain indication method (see further down the discussion of random errors of titrations). 

The kinetics of the binding of 2 -deoxyadcnosine 5 -monophosphate (dAMP) to H2(TMpyP) 52 was studied via the laser Raman temperature jump method. The association- and dissociation-rate constants at 25 "C in pH 6.9 aqueous solution were 1.9 x 10 M s and... 

Schepers, R, inventor, U.S. 5484555 Method for creating a pH jump system, Lever, 1996. 

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