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Affinity Precipitation of Proteins 47
increase in ionic strength, and hence decrease in charge repulsion, by adding
NaCl facilitates precipitation of metal-bound copolymers. At relatively moderate
salt concentrations of 0.4 NaCl, the metal-bound copolymers are precipitated
quantitatively below 25°C (see Fig. 3 ).
5. The poly(VI-co-NIPAM) showed good chelating capacity of metal ions. Cu(II)
and Ni(II) ion binding to poly(VI/NIPAM) increases initially during the first
45–60 min and then levels off toward the equilibrium level (37). The capacities
of poly(VI-NIPAM) (at same VI concentrations in the copolymer) for chelating
Cu(II) ions are slightly more than chelating Ni(II) ions, which can further lead to
different capacities for binding the target protein (15). Our studies have shown
that about two and three imidazole groups co-ordinate with each Cu(II) and Ni(II)
ion, respectively (15). With about two to three imidazole ligands bound to the
metal ion, one could expect binding strength of log K=6–9 (28,30), providing a
significant strength of interaction. At 15 and 25 mol% VI copolymers (i.e., 1.35
and 2.16 μmole VI/mg copolymer, respectively), the Cu(II) and Ni(II) ion content
bound to the copolymers was the same (about 0.6–0.7 μmole/mg copolymer). So
the precipitation efficiency of Cu(II)- and Ni(II)-loaded copolymer at 15 and 25
mol% of VI, respectively for target protein, can be almost the same.
6. Washing of the metal-loaded copolymers three to four times with water (pH
6–7) and by adding 0.4–0.6 M NaCl ensures complete removal of unbound or
100
Relative turbidity (%)
80
60
40
20
0
0 10 20 30 40
Temperature (°C)
Fig. 3. Thermoprecipitation of poly[N-isopropylacrylamide (NIPAM)] and metalloaded
copolymers of poly[vinylimidazole (VI)-NIPAM] from aqueous solution
monitored as turbidity at 470 nm. Maximum turbidity was taken as 100%, and
relative turbidities were calculated from that. Polymer concentration 1 mg/ml.
Poly(NIPAM) precipitation (-•-); Ni(II)-poly(VI-NIPAM) precipitation (--); Cu(II)-
poly(VI-NIPAM) precipitation (--), at different temperatures in presence of 0.4 M
NaCl. VI concentration was 15 and 25 mol% in case of Cu(II) and Ni(II) copolymers,
respectively (reproduced from ref. 2)