Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method.

Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 µM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.

Oriented immobilization of antibodies on a silicon wafer using Si-tagged protein A.

We previously reported a silica-binding protein, designated Si-tag, which can be used to immobilize proteins on silica surfaces. Here, we constructed a fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A for oriented immobilization of antibodies on a silicon wafer whose surface is oxidized to silicon dioxide (silica). The fusion protein, Si-tagged protein A, strongly bound to the silica surface with a dissociation constant of 0.31 nM. Time-of-flight secondary ion mass spectrometry analysis of the silicon wafer coated with Si-tagged protein A, combined with principal component analysis and mutual information, demonstrated that protein A is localized on the outermost surface of the bound protein layer. Immunoglobulin G (IgG) was immobilized both on the silicon wafer coated with Si-tagged protein A and, as a control, directly on the intact silicon wafer via physical adsorption. The silicon wafer coated with Si-tagged protein A bound 30-70% more IgG than the uncoated silicon wafer, whereas the antigen-binding activity was 4- to 5-fold higher for the former, indicating that IgG was functionally immobilized on the silicon wafer via Si-tagged protein A in an oriented manner.