Isoelectric focusing as a method for the characterization of ampholytes : III. Isoelectric points of carrier ampholytes and dissociation constants of some carboxylic acids and alkyl-substituted ammonium ions in sucrose-water, glycerol-water and ethylene glycol-water mixtures
✍ Scribed by W.J. Gelsema; C.L. de Ligny; N.G. van der Veen
- Book ID
- 104145301
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 881 KB
- Volume
- 154
- Category
- Article
- ISSN
- 1873-3778
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✦ Synopsis
Isoelectric points at 25" and 4" of some carrier ampholytes containing different protoQtic groups and dissociation constants at 25" of some carboxylic acids and alkyl-substituted ammonium ions have been determined in water and in sucrosewater, glycerol-water and ethylene glycol-water mixtures.
These data were used to evaluate the errors made in the measurement of isoekctric points of ampholytes (especiahy proteins) by the conventional execution of density-gradient isoelectric focusing.
A correction term, whiclz serves to obtain isoelectric points of proteins in
water from density-gradient isoelectric focusing experiments, is given at 25" and 4" as a function of solvent composition and apparent isoelectric point. The usefulness of this correction term was checked experimentally by performing density-gradient isoekctric focusing experiments on bovine serum albumin in the three solvent systems studied. Its isoelectric point in water at 25" was found to be 4.75 i 0.02. In isoekctric focusing, the pH of the fraction containing the maximum amount of a focused ampholyte is generally called the isoelectric point (pI) of that ampholy3e. This pH value is generally measured electrometrically, i.e., with the aid of a glass electrode, a (satnrati) calomel electrode and a pH meter which is standardized with aqueous standard buffer solutions. However, a pH value determined in that way has no phy&caI n&a&g i;nfess the stipfe is ako an acfueous solution with approximately the same ionic strength as that of the standard buffer solutions used1*2. In that case, pH values can be identifkd very closely (within rO.01 pH unit) with pan.
* For simplicity we neglect here the diEerence between isoprotic and isoelectric point.