| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 93 === The completion of Human Genome Project heralded the beginning of the proteome era. We are urged to develop new technology which shows the potential toward proteomics. By introducing the frontier nano- technology, traditional inhibitor design of targeting enzyme has provided a novel platform for facilitating labeling and purification of protein with high biocomplexity.
Benzene Sulfonamide, a general type of carbonic anhydrase inhibitor combined with both a reactive epoxy group for affinity enhancement on labeling proteins and a thiol tail for anchoring on the gold surface has been synthesized within the scaffold of the tri-functionalized lysine
The functionalized gold nanoparticles GNP-1 was fabricated after grafting compound 1 as ligand on the gold nanoparticles with an average radius of 32nm. Elemental analysis showed that there were about 4000 molecules per particle. Bovine Carbonic Anhydrase II BCA(II) was chosen as a model enzyme. Recognition and affinity binding therefore took place in this micro heterogeneous system containing both GNP-1 and protein solution. GNP-1 was separated from unlabeled protein by centrifugation and analyzed by standard SDS-PAGE and Commasie Blue staining.
Model experiments were carried out at the BCA(II) presence only. In the time- & concentration-dependence experiments, the results showed a 3.3~33 nM range for detection limit for this labeling of BCA(II) and 10 minutes for the labeling to be completed. In the activity-based experiment we denatured the BCA(II) by a pretreatment of heating on 95℃for half an hour. For this denatured BCA(II), GNP-1 displayed higher labeling yield than native one. Selectivity experiment showed no obvious affinity toward four other proteins (BSA, BS-II, RNaseA, and WGA) and proves the recognition occurrence between the designed GNP-1 and BCA(II).
Proteome activity was evaluated by introducing GNP-1 into simple proteins mixture containing BCA(II) and three other proteins. GNP-1 successfully recognized BCA(II) in the mixture and separate them from proteins mixture by centrifugation. And this result was further confirmed by MALTI-TOF for protein identification.
Moreover, ligand structure has been systematically modified as controls for mechanistic investigation during this selective labeling. The result confirms that the selectivity originates from benzene sulfonamide binding to the active site and the epoxide functionality to covalently label the targeted protein.
|
|---|
