PH gradients

The other reactions at the electrodes produce acid (anode) and base (cathode) so that there is a possibiUty of a pH gradient throughout the electrophoresis medium unless the system is well buffered (see Hydrogen-ion activity). Buffering must take the current load into account because the electrolysis reactions proceed at the rate of the current. Electrophoresis systems sometimes mix and recirculate the buffers from the individual electrode reservoirs to equalize the pH. 

Isoelectric Focusing. Isoelectric focusing is a technique used for protein separation, by driving proteins to a pH where they have no mobiUty. Resolution depends on the slope of a pH gradient that can be achieved in a gel. 

Problems associated with gel-to-gel variabiUty have been rectified with the advancement of ampholyte mixtures. One commonly used mixture of ampholytes, called Immobilines, is an improved ampholyte mixture that produces no gradient drift or unequal pH gradient (26) and can be used in a gel matrix reproducibly from one day to the next. 

The possibility of inducing of pH gradients within thin long capillaries with flowing retentive stationary phase based on propylamine or polyethyleneimine was shown. 

The applicability of induced pH gradients for separation of metal ions within anion- and cation-exchange columns was verified. 

Lactoferrin (from human whey). Purified by direct adsorption on cellulose phosphate by batch extraction, then eluted by a stepped salt and pH gradient. [Foley and Bates Anal Biochem 162 296 1987.]... 

Pepsin [9001-75-6] Mf 31,500(human), 6000(hog) [EC 3.4.23.1]. Rechromatographed on a column of Amberlite CG-50 using a pH gradient prior to use. Crystd from EtOH. [Richmond et al. Biochim Biophys Acta 29 453 I958 Huang and Tang, J Biol Chem 244 1085 1969, 245 2189 1970.]... 

When Mitchell first described his chemiosmotic hypothesis in 1961, little evidence existed to support it, and it was met with considerable skepticism by the scientific community. Eventually, however, considerable evidence accumulated to support this model. It is now clear that the electron transport chain generates a proton gradient, and careful measurements have shown that ATP is synthesized when a pH gradient is applied to mitochondria that cannot carry out electron transport. Even more relevant is a simple but crucial experiment reported in 1974 by Efraim Racker and Walther Stoeckenius, which provided specific confirmation of the Mitchell hypothesis. In this experiment, the bovine mitochondrial ATP synthasereconstituted in simple lipid vesicles with bac-teriorhodopsin, a light-driven proton pump from Halobaeterium halobium. As shown in Eigure 21.28, upon illumination, bacteriorhodopsin pumped protons... 

A proton-motive force of approximately —250 mV is needed to achieve ATP synthesis. This proton-motive force, A, is composed of a membrane potential, A P, and a pH gradient, ApH (Chapter 21). The proton-motive force is defined as the free energy difference, AG, divided by S, Paraday s constant ... 

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. 

The methanol gradient from 50 to 60% releases quite a lot of interfering components. Omitting the step gradient does not provide enough selectivity and so the best conditions were obtained with a pH gradient. The experimental conditions are shown in Table 13.1. 

Despite the use of density and pH gradients, cooling and performance in micro-gravitational environments (e.g. the space shuttle) [18], convection and heat dissipation contributed to flow stream instability which was parasitic to the desired separations and limited the utility of this approach. 

Complexes III and IV have Fe-porphyrin prosthetic groups (hemes), complex IV also contains copper atoms which are involved in electron transport. Complexes I, III, and IV use the energy of electron transport to pump protons out of the matrix so as to maintain a pH gradient and an electrical potential difference across the inner membrane required for ATP synthesis (see below and Appendix 3). It is important to remember that all dehydrogenations of metabolic substrates remove two protons as well as two electrons and that a corresponding number of protons are consumed in the final reduction of dioxygen (Figures 5, 6). 

Apart from the passive encapsulation methods, different active entrapment techniques are described in the literature. Nichols and Deamer (1976) prepared liposomes with a pH gradient across the membrane (acidic interior with respect to the external buffer). These liposomes efficiently incorporated several catecholamines added to the external buffer. The same technique has been used to concentrate doxorubicin (DXR) in pH gradient liposomes (Mayer et al., 1986b). 

Mayer, L. D., Hope, M. J., and Cullis, P. R. (1986b). Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient, Biochim. Biophys. Acta. 857. 123-126. 

Nichols, J. W., and Deamer, D. W. (1976). Cathecholamine uptake and concentration by liposomes maintaining pH gradients, Eio-chim. Biophys. Acta, 455, 269-271. 

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.

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