The pH-Gradient

For the practical use of electrofocusing it must be possible to arrange a pH-gradient with suitable properties. The gradient should comprise the desired pH range and other parameters of importance as slope, and conductivity. The pH-gradient may be achieved in two different ways. 

Natural pH-gradients are located and maintained by the electric current itself. The following is a simplified explanation of the natural pH-gradient. 

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In chloroplasts, the value of AT is typically —50 to —100 mV, and the pH gradient is equivalent to about 3 pH units, so that — (2.3 i T/S ) ApH = —200 mV. This situation contrasts with the mitochondrial proton-motive force, where the membrane potential contributes relatively more to bsp than does the pH gradient. 

Figure 16. Chromatographic separation of peptides of a tryptic hydrolysate of the a chain of Hb-St. Claude ona O.9 X O cm column of Chromo-bead resin type P at 37°C. The pH gradient is indicated by the broken line. T-1, T-2, etc. refer to the tryptic peptides and the numbers underneath to the positions in the chain. Several peptides are pure and give satisfactory analyses without further purification.

Figure 20. Chromatographic separation of thermolytic fragments of peptide T-9 isolated from the tryptic digest of the AE-p-chain of Hl Sheperds Bush. The Dowex 1-X2 column uxis 60 cm long and had an internal diameter of 0.6 cm. The pH gradient is indicated by the broken line. The fragments are identified bu their sequences and the positions they occupy in the p-chain. The sequence of the abnormal T-9 is given at the top of the figure.

Reserpine irreversibly inhibits the triphosphatase that maintains the proton gradient and so it depletes neurons of their vesicular store of transmitter. This explains why restoration of normal neuronal function rests on delivery of new vesicles from the cell bodies. Some amphetamine derivatives, including methylenedioxymethamphetamine (MDMA), are also substrates for the transporter and, as a result, competitively inhibit noradrenaline uptake. Another way of inhibiting the transporter is by dissipation of the pH gradient across the vesicular membrane i-chloroamphetamine is thought to act in this way. 

The main problems with early, irreversible MAOIs were adverse interactions with other drugs (notably sympathomimetics, such as ephedrine, phenylpropanolamine and tricyclic antidepressants) and the infamous "cheese reaction". The cheese reaction is a consequence of accumulation of the dietary and trace amine, tyramine, in noradrenergic neurons when MAO is inhibited. Tyramine, which is found in cheese and certain other foods (particularly fermented food products and dried meats), is normally metabolised by MAO in the gut wall and liver and so little ever reaches the systemic circulation. MAOIs, by inactivating this enzymic shield, enable tyramine to reach the bloodstream and eventually to be taken up by the monoamine transporters on serotonergic and noradrenergic neurons. Fike amphetamine, tyramine reduces the pH gradient across the vesicle membrane which, in turn, causes the vesicular transporter to fail. Transmitter that leaks out of the vesicles into the neuronal cytosol cannot be metabolised because... 

The half-wave potentials of (FTF4)Co2-mediated O2 reduction at pH 0-3 shifts by — 60 mV/pH [Durand et ah, 1983], which indicates that the turnover-determining part of the catalytic cycle contains a reversible electron transfer (ET) and a protonation, or two reversible ETs and two protonation steps. In contrast, if an irreversible ET step were present, the pH gradient would be 60/( + a) mV/pH, where n is the number of electrons transferred in redox equilibria prior to the irreversible ET and a is the transfer coefficient of the irreversible ET. The —60 mV/pH slope is identical to that manifested by simple Ee porphyrins (see Section 18.4.1). The turnover rate of ORR catalysis by (ETE4)Co2 was reported to be proportional to the bulk O2 concentration [Collman et ah, 1994], suggesting that the catalyst is not saturated with O2. 

Said et al. [78] directly measured the acid microclimate on the surface of gastrointestinal tract (GIT) epithelial cells (intact with mucus layer) in rats. The pH on the apical (donor) side of the cells varied from 6.0 to 8.0, while the pH on the basolateral (acceptor) side was 7.4. Furthermore, the pH gradient between... 

In iso-pH serum protein- and surfactant-free solutions, the concentration of the sample in the acceptor wells cannot exceed that in the donor wells. With gradient-pH conditions, this limitation is lifted. At very long times, the concentrations in the donor and acceptor chambers reach equilibrium values, depending on the pH gradient... 

Antonenko et al. [540] considered pH gradients forming in the UWL under bulk solution iso-pH conditions. They elegantly expanded on the buffer effect model and made it more general by considering multicomponent buffer mixtures. Direct measurements of the pH gradients (using wire-coated micro-pH electrodes) near the membrane-water interface were described. 

The third factor is the behavior of primary chemicals in the soil at different pH conditions. The chemistry in the system is governed by the pH gradients across the soil mass. Knowledge of the behavior of the primary chemicals in different pH environments is necessary for a better understanding of the efficiency and to enable a decision to be made on the required processing conditions and time. 

Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Mitochondria do three things oxidize substrates, consume oxygen, and make ATP. Uncouplers prevent the synthesis of ATP but do not inhibit oxygen consumption or substrate oxidation. Uncouplers work by destroying the pH gradient. The classic uncoupler is dinitrophenol (DNP). This phenol is a relatively strong acid and exists as the phenol and the phenolate anion. 

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