38
chapter 3
Protein Isolation and Determination of Amino Acid Sequence
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F IG U R E 3-2
Gel filtration for separation of solutes by size. Solute molecules smaller than the diameter of the pores of the gel
particles enter them and are retained for a longer time. Larger solute molecules cannot penetrate the pores of the gel
particles and are eluted off the column first.
and pH. The resins are inert polymers to which ionizable
groups have been attached; resins with negative charges
are cation exchangers, and those with positive charges are
anion exchangers. Two ion exchange resins frequently
used in protein purification are diethylaminoethylcel-
lulose (DEAE-cellulose, an anion exchanger) and car-
boxymethylcellulose (CM-cellulose, a cation exchanger).
A protein with a net positive charge at a given pH will com-
bine with the negative groups of a cation exchange resin,
and its flow will be retarded; a protein with a net nega-
tive charge will migrate through the cation exchange resin
unimpeded. Cationic proteins in a mixture going through
the column will compete with one another for binding to
the negatively charged groups of the resin. The relative
migration rates of different molecules depend on three
factors: their individual affinities for the charged sites on
the resin, the degree of ionization of the functional groups
attached to the resin, and the chemical properties and con-
centrations of competing low-molecular-weight ions, e.g.,
potassium and sodium. For example, NaCl at the appropri-
ate concentration can displace a cationic protein through
competition between sodium ions (Na+) and the positively
charged groups of the protein for the negatively charged
sites on the resin (Figure 3-3). Changing the pH of the elut-
ing buffer can also bring about desorption of proteins from
the resin through neutralization of the ionizable R-groups
of amino acid residues. Decreasing pH will elute proteins
in the order of their decreasing isoelectric point values
during anion exchange chromatography.
Affinity chromatography
takes advantage of specific
affinities between protein molecules and analogues of bi-
ological molecules that are covalently bound to the column
