Donald M. Kurtz, Ph.D. , University of Texas (San Antonio)
During the first year, we developed methods for incorporating ZnPPIX into Bfr. All manipulations were conducted in low light conditions, and the ZnPPIX-Bfr preparations were stored in the dark. Bfr was overexpressed in a special E. coli strain containing a plasmid which expresses a heme receptor in its outer membrane. We found that use of this strain resulted in greatly increased uptake of exogenously added ZnPPIX compared to E. coli strains lacking the heme receptor plasmid. The internalized ZnPPIX was then incorporated, apparently spontaneously, into the overexpressed Bfr. The ZnPPIX-Bfr was purified in good yield from the cell extracts by anion-exchange and gel filtration chromatographies. UV-vis absorption and protein analyses showed that ZnPPIX was quantitatively incorporated into Bfr, i.e., 12 ZnPPIX per Bfr 24-mer with little or no heme contamination. Using this same expression protocol, ZnPPIX was not incorporated into a site-directed Bfr variant known to be incapable of binding heme, but which otherwise forms a stable 24-mer, indicating that the ZnPPIX occupies the native heme binding sites. The UV-vis absorption spectra of ZnPPIX-Bfr closely resemble those of ZnPPIX incorporated into heme binding sites of other proteins. The as-isolated ZnPPIX-Bfr typically contained ~30 non-heme iron atoms per Bfr 24-mer. As described below, this relatively low non-heme iron content did not interfere with subsequent nanoparticle incorporation or photochemistry. As a test of photochemical activity, we found that irradiation of the ZnPPIX-Bfr with visible light from a tungsten halogen projector lamp in the presence of the sacrificial electron donor, triethanolamine, caused reduction of the redox dye, methyl viologen, over the course of several minutes. No photoreduction of methyl viologen occurred in the absence of either visible light irradiation or the sacrificial electron donor.
We then adapted published procedures used with other proteins to incorporate platinum NPs into the internal cavity of ZnPPIX-Bfr. The ZnPPIX-Bfr was incubated with excess PtCl62- or PtCl42-, which diffuses into the interior cavity of the protein. Sodium borohydride was then added to reduce the internalized platinum ions to Pt(0). Excess reagents were removed by passage over a small size-exclusion column. Numerous experiments were conducted in which Pt salt-to-protein ratio, borohydride-to-Pt ratio, pH, temperature, and reaction time were varied in order to optimize conditions for Pt NP incorporation. Under the optimized conditions (pH 7, room temperature, ~1600 Pt/Bfr 24-mer and ~0.5 mol borohydride/mol Pt) transmission electron microscopy showed that most of the Bfr 24-mers enclosed 3- to 5-nm Pt NPs, which were not present in protein that had not been exposed to platinum. Few or no NPs were evident outside the Bfr 24-mers. UV-vis absorption spectra showed that the ZnPPIX was retained in the Bfr preparations containing the Pt NPs. During the remainder of the grant period, we will attempt to detect and optimize photochemically generated H2 by gas chromatography.
This PRF-supported project has jump-started an entirely new and exciting research direction in my laboratory involving the construction of protein scaffolds for novel photochemical transformations. For example, I have obtained a grant to explore the use of Bfr as a photochemical Fenton-iron delivery system in cancer therapy. An undergraduate in my laboratory has succeeded in incorporating palladium nanoparticles into ZnPPIX-Bfr, which we plan to use for photo-driven hydrogenation reactions. A graduate student, Emily Clark, has carried out the PRF-supported research described above, and is completing the second year of her Ph.D. degree program. She recently presented a poster on this research at the 15th International Conference on Bioinorganic Chemistry in Vancouver, BC, August 7-12, 2011. The PRF grant has allowed Emily the luxury of exploring this new avenue of research in my lab. It seems likely that the novelty, interdisciplinary nature, and potential practical applications of this research will broaden her career options.