Use of bulky ligands (BLs) in the synthesis of metal nanoparticles (NPs) gives smaller core sizes, sharpens the size distribution, and alters the discrete sizes. For BLs, the highly curved surface of small NPs may facilitate growth, but as the size increases and the surface flattens, NP growth may terminate when the ligand monolayer blocks BLs from transporting metal atoms to the NP core. Batches of thiolate-stabilized Au NPs were synthesized using equimolar amounts of 1-adamantanethiol (AdSH), cyclohexanethiol (CySH), or n-hexanethiol (C6SH). The bulky CyS- and AdS-stabilized NPs have smaller, more monodisperse sizes than the C6S-stabilized NPs. As the bulkiness increases, the near-infrared luminescence intensity increases, which is characteristic of small Au NPs. Four new discrete sizes were measured by MALDI-TOF mass spectrometry, Au(30)(SAd)(18), Au(39)(SAd)(23), Au(65)(SCy)(30), and Au(67)(SCy)(30). No Au(25)(SAd)(18) was observed, which suggests that this structure would be too sterically crowded. Use of BLs may also lead to the discovery of new discrete sizes in other systems.

Human insulin was labeled with fluorescein isothiocyanate (FITC) and fully characterized to yield four distinct insulin-FITC species. High-performance liquid chromatography and electrospray mass spectrometry were used to determine the extent and location of fluorescein conjugation. By changing the reaction conditions (i.e., pH, time, and FITC/insulin ratio) the selectivity of the fluorescein conjugation was altered, and all conjugates could be separated. The isolated species of insulin-FITC were labeled at the following residues: A1(Gly), B1(Phe), A1(Gly)B1(Phe), and A1(Gly)B1(Phe)B29(Lys). All four insulin-FITC conjugates were then used to develop fluorescence polarization binding assays with monoclonal and polyclonal anti-insulin antibodies. The assay sensitivity differed between the conjugates depending on the site of modification (B1 > A1 > A1B1 > A1B1B29). Also, the type of antibody used had an important role in the binding of insulin-FITC conjugates. Finally, for the first time the biological activity of the four conjugates was demonstrated by an autophosphorylation assay. The positional substitution dramatically affected the biological activity, confirming insights into the residues responsible for the insulin binding region. The B1 conjugate was found to retain almost all biological activity while the A1 and A1B1 conjugates had approximately 10 times lower activity. The trisubstituted species (labeled at A1, B1, and B29) was determined to be least active.

An affinity chromatography system has been developed for the separation of recombinant fusion proteins based on the Ca(2+)-dependent binding of calmodulin (CaM) to the drug phenothiazine. Specifically, in the presence of Ca2+, a recognition site for phenothiazine is exposed on calmodulin, allowing the binding of this drug to CaM. Upon removal of Ca2+ with EGTA, the conformation of calmodulin changes, and the phenothiazine--CaM complex dissociates. This Ca(2+)-dependent binding has been exploited in the development of a fusion tail approach for the affinity purification of recombinant proteins and peptides. Protein A (ProtA) was employed as a model protein to demonstrate the advantages of this approach. In particular, the developed affinity chromatography system was used to isolate several ProtA--CaM fusion proteins. These recombinant fusion proteins were expressed in Escherichia coli and Saccharomyces cerevisiae from appropriately designed plasmids. Four different plasmids (two each for the bacteria and yeast) were used that encoded the fusion of CaM to the immunoglobulin-binding portion of protein A. After expression of the fusion protein, the crude cell lysates were loaded onto the phenothiazine affinity column in the presence of a Ca(2+)-containing buffer. Upon elution with an EGTA buffer, the ProtA--CaM fusion protein was purified, as confirmed by SDS-PAGE electrophoresis and Western blot analysis.

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