Elsevier

Analytical Biochemistry

Volume 90, Issue 2, 15 October 1978, Pages 633-643
Analytical Biochemistry

Hindered migration of amino acids toward their isoelectric positions in gel electrofocusing, and facilitation of the migration at high ionic strength

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Abstract

Amino acids with a largepI -pKp difference are known to be poor carrier ampholytes in electrofocusing, exhibiting isoelectric zones with poor conductivity across as many as 4 pH units. Accordingly, radioactive amino acids of this type, e.g., glycine, are found to be distributed over the entire pH gradient formed by Ampholine in electrofocusing gels, while radioactive amino acids like histidine or glutamic acid with small pI - pKp differences form single peaks at or near their pI's. When poor carrier ampholyte amino acids are subjected to gel electrofocusing in 0.1 m KCl, their distribution sharpens into single peaks, at or near the pI, indistinguishable from those of the good carrier ampholyte amino acids. At an intermediate stage of peak coalescence of the original broad distributions of poor carrier ampholyte amino acids, in 0.01 m KCl, acidic and basic peaks of amino acid can be observed, possibly analogous to acidie and basic distributions previously observed with labeled Ampholine. The rate of peak coalescence of anionic amino acids seems higher than that of the cationic species. The mechanism by which high ionic strength facilitates the condensation of poor carrier ampholyte amino acids at their pI remains unknown. Possibly, the current within zones of poor carrier ampholyte amino acids is insufficient, or poor carrier ampholyte amino acids are not sufficiently charged, to allow for electrophoretic migration of the bulk of loaded amino acid to its isoelectric position, unless the current density is increased by electrofocusing at high ionic strength. Alternatively, 0.1 m KCl may interfere with electrovalent interactions between amino acids and isoelectric carrier ampholyte zones, analogous to the action of urea in preventing the interaction between polyanions and carrier ampholytes.

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