Ampholytes ionic strength

J. THE CARRIER AMPHOLYTES, IONIC STRENGTH AND INFLUENCE ON SOLUBILITY OF PROTEINS... 

Salt should be present in a sample only if it is an absolute requirement. Carrier ampholytes contribute to the ionic strength of the solution and can help to counteract a lack of salts in a sample. Small samples (1 to 10 /zL) in typical biochemical buffers are usually tolerated, but better results can be obtained with solutions in deionized water, 2% ampholytes, or 1% glycine. 

It is of interest to note that equation (21) requires the solubility of a dipolar ampholyte molecule to increase in the presence of neutral salts the solubility of a neutral, uncharged molecule on the other hand decreases with increasing ionic strength of the medium (cf. p. 147). The fact that the solubility of glycine and other aliphatic amino-aeids increases under these conditions constitutes, as already mentioned, evidence for their dipolar structure. 

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... 

The direction and also the extent of migration of ampholytes are thus pH dependent and buffers ranging from pHl to pH 11 can be used to produce the required separations. The correct buffer, that is the one which has the most apprporiate value of pH and ionic strength for the particular separation in hand, is found by experimentation. 

At or in the proximity of their pi values proteins exhibit a minimum total charge and reduced solubility in the electrolyte solution [350,370,385]. This increases the probability of aggregation, and is further enhanced by the low ionic strength of the ampholyte buffer. Under these conditions protein precipitation results from hydrophobic interactions. These interactions can be suppressed by addition of additives such as ethylene glycol (10-40 %), non-ionic or zwitterionic surfactants (1-4 %), or sorbitol to the ampholyte buffer. 

The ions under consideration are assumed to be freely moving and charges of opposite sign are assumed not to be separated. The concept is much used in connection with the electrophoresis of proteins. The solubility of proteins depends partly on the ionic conditions of the environment. In salt solutions these ionic conditions can be discussed satisfactorily with the ionic strength concept, but not in ampholyte solutions. In spite of the fact that the ionic strength is low, the carrier ampholytes at their isoelectric points contribute to the ionic cloud surrounding the dissolved protein molecules. Thus they also contribute to the solubility and stability conditions of the protein molecule. 

The terms Ca and Ct are defined by equations (3 9a) and (3-9) respectively. The hydrogen and hydroxyl ion activity terms have been placed in the equation to emphasize that these are assumed to be the measured quantities derived from the pH measurements. Whilst it is correct to use the hydrogen ion activity in equation (3-22), the hydrogen ion concentration is required for equation (3-22a). It is apparent, therefore that mixed constants would result from the solution of equation (3 -22). A further complication is the ionic strength which, in this case, cannot be calculated directly from the stoicheiometric concentration used, for example, in equation (3-19). To obtain the thermodynamic values, K and KI, the activity functions must be calculated from estimates of the ionic strength. How this can be achieved will now be described for dibasic acids followed by a similar method for ampholytes and diacidic bases. 

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