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of the ChT-polymer complex was studied toward anionic, neutral, and cationic
substrates. Because the polymer-protein interaction is based on electrostatics, we
speculated that the negative charge of N-(succinyl)-
L
-phenylalanyl-p-nitroanilide
could influence the inhibition efficiency of ChT (Fischer et al. 2002, 2003). Indeed,
there was an enhanced activity of ChT against the cationic substrate, but a decrease
in activity in the case of the anionic substrate. These results imply that the negatively
charged polymer plays a role in recruiting the substrates to the active site of the protein.
Similarly, this amphiphilic polymer micelle was also used to disrupt the complex
between cytochrome c (Cc) and cytochrome c peroxidase (CcP; Sandanaraj,
Bayraktar et al. 2007). In this case, we found that the polymer modulates the redox
properties of the protein upon binding. The polymer binding exposes the heme
cofactor of the protein, which is buried in the protein and alters the coordination
environment of the metal. The exposure of heme was confirmed by UV-vis, CD
spectroscopy, fluorescence spectroscopy, and electrochemical kinetic studies. The
rate constant of electron transfer (k
0
) increased by 3 orders of magnitude for the
protein-polymer complex compared to protein alone. To establish that the
polymer micelle is capable of disrupting the Cc-CcP complex, the polymer
micelle was added to the preformed Cc-CcP complex. The k
0
observed for this
complex was the same as that of the Cc-polymer complex, which confirms that
the polymer micelle is indeed capable of disrupting the Cc-CcP complex.
2.3.3. Protein Sensing
Because these polymeric amphiphiles are capable of binding to proteins, we envi-
saged the possibility of detecting metalloproteins using fluorescence quenching.
Conjugated polymers containing charged functionalities have been utilized as fluor-
escent sensing elements (Chen et al. 1999; Fan et al. 2002; Wilson et al. 2003; Kim
et al. 2005), because the charges can bind to the complementary charges in the pro-
teins, whereas the inherent fluorescence of the polymer could potentially be affected
by the cofactors in metalloproteins. Porphyrin-based or heavy metal based cofactors
in metalloproteins can accept energy or electrons from the excited state of the conju-
gated polymer chromophores. Conjugated polymers have been shown to be effective
in sensing metalloproteins, but it has also been shown that fluorescence changes can
be observed with nonmetalloproteins because of conformational or aggregation
changes in conjugated polymers. We hypothesized (Sandanaraj et al. 2006) that uti-
lizing the amphiphilic homopolymers with pendant chromophores (Chart 2.9) would
provide all the advantages of conjugated polymers, while being selective only to
metalloproteins. This is because even when the protein binding could cause gross
conformational changes in the polymer, the emission properties of a pendant chromo-
phore in a nonconjugated polymer should remain unaffected. Therefore, the only
feature that this polymer would report is whether there is a metalloprotein cofactor
that can accept energy or electrons from its excited state. We have shown that such
a strategy indeed results in selective sensors for metalloproteins (Fig. 2.9).
The approach above could distinguish metalloproteins from nonmetalloproteins.
However, it is interesting to provide an approach that distinguishes among the
