Biomedical Engineering Reference
In-Depth Information
FIGURE 11.7
X-ray crystal structure of the GST Dimer (PDB code 12GS). (From Oakley, A. J., LoBello, M., Nuccetelli, M.,
Mazzetti, A. P., and Parker, M. W. (1999). The ligandin (non-substrate) binding site of human pi class glutathione
transferase is located in the electrophile binding site (H-site).
J. Mol. Biol.
291: 913.).
enlarge the pores, and stabilized via thermal oxidation. To prepare surfaces for immobi-
lizing GST, PSi samples were first treated with a solution of 3-aminopropyltriethoxysi-
lane (APTS), followed by a solution of glutaraldehyde. This process yields a surface
populated with reactive aldehyde groups, capable of trapping amines on the protein
(from the amino terminus and lysine side-chains) as imines. Following immobilization
of GST, measurement of the enzyme reaction yield on the chip compared to measure-
ments made with free enzyme in solution allowed for the amount of enzyme immobi-
lized to be determined. As expected, we found that the amount of enzyme immobilized
increased with increasing depth of the PSi layer (Figure 11.8). Furthermore, the observed
immobilization capacity correlated well with a simple geometric model of the PSi matrix
in which the pores are approximated as cylinders (Figure 11.9). These results established
that immobilization of biomolecules in PSi does indeed proceed in a predictable manner
and set the stage for further characterization of the enzymatic and optical performance
of multilayer films.
While GST retained its ability to function inside single-layer (single-porosity) PSi films,
would the same be true of multilayer films? To examine this question, we prepared
/2
microcavity devices consisting of 28 layers of alternating 71 and 84% porosity,
31
and
immobilized GST as before. After carefully characterizing the kinetic parameters for free
GST in solution as a function of concentration, enzymatic activity of the microcavity-
immobilized GST was assessed. Perhaps unsurprisingly, the activity of the immobilized
GST was between two- and fourfold lower than that in solution (Table 11.1). Several effects
potentially contribute to this observation. First, the immobilization chemistry employed in
these studies is not regiospecific: any amine on the surface of the protein has an equiva-
lent probability of reacting with an aldehyde on the PSi surface. Thus, it is possible for the
enzyme to be attached in an orientation that blocks access to the substrate-binding site.
