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304 Heegaard et al.
1. Introduction
Very highly efficient separations that are reminiscent of the capabilities of
high-performance liquid chromatography (HPLC) but do not require reversed
phase conditions can be achieved by capillary electrophoresis (CE). Highvoltage
electrophoresis in solution in sub-millimetre diameter quartz tubes
was introduced in the end of the 1980s (1–7), and CE has been used much
since, especially for characterizing small molecules. The technique has unique
capabilities, e.g. for separating impurities and enantiomers using simple, short
procedures. Also, the potential for automation has been extremely successfully
combined with parallel processing and laser-induced fluorescence detection in
DNA-sequencing where CE has become the most important separation method.
As for other biological macromolecules, the chemically more complex polypeptides
and proteins have proved to be challenging to analyse using CE. Despite
this, CE offers unique possibilities for functional characterization of proteins
(8) by exploiting and characterizing binding interactions in which the protein
takes part. Protein CE involving reversible molecular interactions, known as
protein affinity CE (ACE), is the topic of this chapter.
Proteinaceous biomolecules are complicated analytes because they are not
always structurally or conformationally homogeneous, since they may be
composed of distinct subdomains with widely different properties and because
they are often only available in limiting amounts which hampers method development.
The goal of functional characterization of proteins is typically to
understand their role at their point of origin, i.e., under physiological conditions
or conditions as near physiological as possible. This chapter is intended to
give a discussion of CE methods for this purpose from a practical perspective
with an emphasis on the most critical factors for successful analyses and
with application examples illustrating various types of information garnered
from CE-based affinity studies. The literature is not reviewed comprehensively.
A number of recent publications may be consulted for more systematic reviews
of interaction applications and the theory of CE (9–20).
2. Objectives and Limitations
There are no simple and universal rules as to the size, isoelectric point, amino
acid composition, conformational characteristics, solubility or other molecular
features that predict whether CE investigations of proteins are going to be
applicable and how they should be carried out. Some generalizations, however,
can be made: If a protein is small, structurally homogeneous, conformationally
stable, globular, well-soluble and negatively charged, then chances are good
that characterizing this particular protein by CE separations in buffers near
physiological pH and ionic strength values will be feasible. The objectives of
using molecular interactions in CE can be different: discovery and mapping of