ABSTRACT
A straightforward indicator-displacement assay (IDA) has been developed for the quantitative analysis of ATPâADP conversion. The IDA relies on the use of gold nanoparticles passivated with a monolayer of thiols terminating with a 1,4,7-triazacyclononane (TACN)·Zn(2+) head group. The analytes ATP and ADP compete to a different extent with a fluorescent probe for binding to the monolayer surface. In the presence of ATP the fluorescent probe is free in solution, whereas in the presence of ADP the fluorescent probe is captured by the nanoparticles and its fluorescence is quenched. The linear response of the fluorescence signal towards different ratios of ATP : ADP permitted the detection of protein kinase activity simply by adding aliquots of the enzyme solution to the assay solution followed by measurement of the fluorescent intensity. The assay poses no restrictions on the target kinase nor does it require labeling of the kinase substrate. The assay was tested on the protein kinases PIM-1 and Src and validated through a direct comparison with the classical radiometric assay using the [γ-(32)P]-labeled ATP.
Subject(s)
Enzyme Assays/methods , Fluorescent Dyes/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Protein Kinases/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Protein Kinases/chemistry , Proto-Oncogene Proteins c-pim-1/chemistry , Proto-Oncogene Proteins c-pim-1/metabolism , Spectrometry, Fluorescence , src-Family Kinases/chemistry , src-Family Kinases/metabolismABSTRACT
Histidine-containing peptides self-assemble on the surface of monolayer protected gold nanoparticles to form a catalytic system for transesterification reactions. Self-assembly is a prerequisite for catalysis, since the isolated peptides do not display catalytic activity by themselves. A series of catalytic peptides and substrates are studied in order to understand the structural parameters that are of relevance to the catalytic efficiency of the system. It is shown that the distance between the His-residue and the anionic tail does not affect the catalytic activity. On the other hand, the catalytic His-residue is sensitive to the chemical nature of the flanking amino acid residues. In particular, the presence of polar Ser-residues causes a significant increase in activity. Finally, kinetic studies of a series of substrates reveal that substrates with a hydrophobic component are very suitable for this catalytic system.
Subject(s)
Histidine/chemistry , Nanoparticles/chemistry , Peptides/chemistry , Catalysis , EsterificationABSTRACT
Catalytically active peptide-nanoparticle complexes were obtained by assembling small peptide sequences on the surface of cationic self-assembled monolayers on gold nanoparticles. When bound to the surface, the peptides accelerate the transesterification of the p-nitrophenyl ester of N-carboxybenzylphenylalanine by more than 2 orders of magnitude. The gold nanoparticle serves as a multivalent scaffold for bringing the catalyst and substrate into close proximity but also creates a local microenvironment that further enhances the catalysis. The supramolecular nature of the ensemble permits the catalytic activity of the system to be modulated in situ.
