Affinity Chromatography - Department of Molecular and Cellular ...

DNA binding proteins
HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow
DNA binding proteins form an extremely diverse class of proteins sharing a single characteristic,
their ability to bind to DNA. Functionally the group can be divided into those
responsible for the replication and orientation of the DNA such as histones, nucleosomes
and replicases and those involved in transcription such as RNA/DNA polymerases, transcriptional
activators and repressors and restriction enzymes. They can be produced as
fusion proteins to enable more specific purification (see page 42), but their ability to bind
DNA also enables group specific affinity purification using heparin as a ligand. Heparin is
a highly sulphated glycosaminoglycan with the ability to bind a very wide range of
biomolecules including:
• DNA binding proteins such as initiation factors, elongation factors, restriction
endonucleases, DNA ligase, DNA and RNA polymerases.
• Serine protease inhibitors such as antithrombin III, protease nexins.
• Enzymes such as mast cell proteases, lipoprotein lipase, coagulation enzymes,
superoxide dismutase.
• Growth factors such as fibroblast growth factor, Schwann cell growth factor,
endothelial cell growth factor.
• Extracellular matrix proteins such as fibronectin, vitronectin, laminin,
thrombospondin, collagens.
• Hormone receptors such as oestrogen and androgen receptors.
• Lipoproteins.
The structure of heparin is shown in Figure 33. Heparin has two modes of interaction with
proteins and, in both cases, the interaction can be weakened by increases in ionic strength.
1. In its interaction with DNA binding proteins heparin mimics the polyanionic structure
of the nucleic acid.
2. In its interaction with coagulation factors such as antithrombin III, heparin acts as an
affinity ligand.
(A)
(B)
O
O
COO –
OH
O
H 2 COR 1
OH
O OH O
HNR
COO – 2
O
OH
OR 1
Fig. 33. Structure of a heparin polysaccharide consisting of alternating hexuronic acid (A) and D-glucosamine residues
(B). The hexuronic acid can either be D-glucuronic acid (top) or its C-5 epimer, L-iduronic acid (bottom).
R 1
= -H or -SO 3–
, R 2
= -SO 3
–
or -COCH 3
.
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