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We report a rapid colorimetric assay to detect protein phosphatase (PP) activity based on the controlled assembly and disassembly of gold nanoparticles (AuNPs) via Zn(II)-specific coordination in the presence of His6-tagged phosphopeptides. Among divalent metal ions including Ni(II), Cu(II), Co(II), Mg(II), Mn(II), and Zn(II), only Zn(II) triggered a strong association between phosphopeptides with hexahistidine at a single end and nitrilotriacetic acid (NTA)-modified AuNPs (21.3 nm in core diameter), leading to the self-assembly of AuNPs and consequently changes in color of the AuNP solution. In contrast, unphosphorylated peptides and His6-deficient phosphopeptides did not change the color of the AuNP solution. As a result, protein phosphatase 1 (PP1) activity and its inhibition were easily quantified with high sensitivity by determining the extinction ratio (E520/E700) of colloidal AuNPs. Most importantly, this method was capable of detecting protein phosphatase 2A (PP2A) activity in immunoprecipitated plant extracts. Because PPs play pivotal roles in mediating diverse signal transduction pathways as primary effectors of protein dephosphorylation, we anticipate that our method will be applied as a rapid format method to analyze the activities of various PPs and their inhibition.

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We report bioluminescence analysis of matrix metalloproteinase (MMP) activity in biological substances using a surface-bound luciferase probe. Intein-fused luciferase protein enables site-specific biotinylation of luciferase in the presence of N-terminus cysteine-biotin via intein-mediated splicing process, resulting in a strong association with high bioluminescence signal onto a NeutrAvidin-coated surface. When the peptide substrate for MMP-7 was inserted into a region between luciferase and intein, the biotinylated probe detected MMP-7 activity by cleaving the peptide, and surface-induced bioluminescence signal was strongly reduced in the MMP-secreted media or mouse tissue extracts, compared with that in MMP-deficient control set. Our approach is anticipated to be useful for generating biotinylated proteins and for their applications in diagnosing MMP activity in human diseases.

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