Ampholytes, buffer capacity

Shiau et al. [73] directly measured the microclimate pH, pHm, to be 5.2-6.7 in different sections of the intestine (very reproducible values in a given segment) covered with the normal mucus layer, as the luminal (bulk) pH, pH/, was maintained at 7.2. Good controls ruled out pH electrode artifacts. With the mucus layer washed off, pHm rose from 5.4 to 7.2. Values of pHfo as low as 3 and as high as 10 remarkably did not affect values of pHm. Glucose did not affect pHm when the microclimate was established. However, when the mucus layer had been washed off and pHm was allowed to rise to pHfo, the addition of 28 mM glucose caused the original low pHm to be reestablished after 5 min. Shiau et al. [73] hypothesized that the mucus layer was an ampholyte (of considerable pH buffer capacity) that created the pH acid microclimate. 

Figure 9.5 Generation of a pH gradient by ampholytes within a capillary flanked by an acid as anodic solution and base as cathodic solution. Ampholyte solutions are composed of high numbers of low-molecular weight amphoteric electrolytes (from which the name is derived) with slightly different pi values. Because ampholytes possess buffering capacity, they maintain a pH value in the specific area occupied by the different molecular species. The sample, which is also amphoteric, focuses in between ampholytes with higher and lower pi. To achieve resolution, there must be at least one ampholyte with a pi intermediate to the two sample components of interest.

The first practical IEF experiments were carried out with the use of synthetic molecules, called carrier ampholytes, to generate the pH gradients.1,26 Carrier ampholytes are amphoteric electrolytes that carry both current and buffering capacity. Much of the early theoretical activity in electrofocusing dealt with the properties required of carrier ampholytes and is more or less irrelevant to a current discussion.1,3,9 Different varieties of... 

Figure 11.2 shows the pH gradients calculated for mixtures of eight carrier ampholyte species possessing pi differences of 0.05 and 0.10 pH units. The individual species focus at their pi values, where their buffering capacity is low. A linear... 

The first studies of carrier ampholytes were conducted using amino acids and dipeptides, but these species did not work well because their pK values for the amino and carboxylate groups are too far removed from their pi values. After they were prefocused, these species had very low buffering capacity. Good... 

Carrier ampholyte-based IEF methods are commonly used in situations where very high resolution of proteins according to their pi values is not required. Several problems exist with the use of carrier ampholytes that limit their resolving power. These include the low and uneven ionic strength that results in smearing of the most abundant proteins in the sample, the uneven buffering capacity and conductivity, the unknown chemical environment, a low sample loading capacity, and a... 

Figure 5-4 Schematic of an lEF procedure, i, A homogeneous mixture of carrier ampholytes, pH range 3 to 10, to which proteins A, B, and C with pi 8, 6, and 4, respectively, were added. 91, Current is applied and the carrier ampholytes rapidly migrate to the pH zones where the net charge is zero (the pi value). Ill, The proteins A, B, and C migrate more slowly to their respective pi zones where migration ceases.The high buffering capacity of the carrier ampholyte creates stable pH zones in which each protein may reach its pi.

The pH gradient in the gel is created by using proprietary mixtures of small ampholytes with good buffering capacity. 

Due to the high buffering capacity of the ampholytes, the pH gradient is stable even in the presence of larger concentrations of analytes. However, sometimes the gradient starts to drift over time, often towards the cathode. This compromises the performance of lEF. 

The pH-gradient must have sufficient buffer capacity to be able to dictate a pH-gradient, so that the buffering properties of the sample proteins or other sample ampholytes do not change the course of the pH-gradient. Also, the carrier ampholytes must be sufficiently soluble in water to reach the necessary buffering capacity. 

Everywhere in the pH-gradient there must be a sufficient buffering capacity to prevent the protein components of the sample specimen from affecting the pH. In other words, each carrier ampholyte must have sufficient buffering capacity at its isoelectric point. 

If the charge Q is plotted against pH, the result is identical with the titration curve of the ampholyte. The buffering capacity can thus be expressed ... 

Figure 3. The buffering capacity of a carrier ampholyte as a function of pi — pK. The intercept with the ordinate axis is the limiting buffering capacity. Also, when pi — pK is 1 pH unit, of the limiting capacity is still retained. The curves shows that carrier ampholytes should have the property of being isoelectric between two closely spaced pK values (Svensson, 2).

Figure 4. Titration curves of glutamic acid, glycine, histidine, and lysine. The three amino acids whose titration curves sharply cut the zero-charge-horizontal-line also have a net charge apart from zero in the vicinity of the pH where they cut the zero line, i.e., the pi. That means that they have buffering capacity and conductance in the neighborhood of the isoelectric point and therefore are useful as carrier ampholytes. Glycine, on the other hand, with its extended horizontal part of the curve is not suitable. (Svensson, 2.)...

The linear part of the pH-gradient terminates at about pH 3.5 on the acid side and about 9.5 on the basic side. Toward the ends of the gradient, the number of different ampholyte molecules diminishes. The buffering capacity therefore becomes low there. However, in these pH ranges there are other good carrier ampholytes available. By adding, for exam-... 

After labeling, 2xlysis buffer [8 M urea, 4% (w/v) CHAPS, 2% (v/v) carrier ampholytes, 2% (w/v) DTT] can be added to the samples in a l-tl dilution for lEF. Combining the three samples to be separated in one lEE strip or tube gel results in a total protein concentration of 150 pg per gel (50 pg Cy3-i-50pg Cy5-t50pg Cy2 labeled). From here methods are virtually identical to classical 2-DE. A peculiarity is that glass cassettes should have low intrinsic fluorescence capacity, since the gel will be scanned still assembled between the plates. 

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