High-sensitivity miniaturized immunoassays for tumor necrosis factor alpha using microfluidic systems.

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels.

A comprehensive proteome map of growing Bacillus subtilis cells.

The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology. DNA arrays were used to calculate the number of genes transcribed in growing cells. From the 4100 B.

UV-A/blue-light inactivation of the 'metal-free' hydrogenase (Hmd) from methanogenic archaea.

H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed 'metal-free' hydrogenase does not contain iron-sulfur clusters or nickel and thus differs from [Ni-Fe] and [Fe-Fe] hydrogenases, which are all iron-sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit, but the iron was considered to be a contaminant as none of the catalytic and spectroscopic properties of the enzyme indicated that it was an essential component.

Measurement and ab initio calculation of CSA/dipole-dipole cross-correlated relaxation provide insight into the mechanism of a H2-forming dehydrogenase.

Using transferred cross-correlated relaxation and DFT calculations, the conformation of the relevant conformation of N5,N10-methylenetetrahydromethanopterin, a reaction intermediate bound to the 80 kD H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase is determined. The conformation of the intermediate differs from the free form in solution and makes the reaction mechanism plausible.

Catalytic Mechanism of the Metal-Free Hydrogenase from Methanogenic Archaea: Reversed Stereospecificity of the Catalytic and Noncatalytic Reaction.

By activation of the hydrogen acceptor, the metal-free hydrogenase from methanogenic archaea catalyzes the reduction of methenyl tetrahydromethanopterin with H . According to NMR spectroscopic analysis of the conformation of the hydrogen acceptor in solution and of the stereospecificity of the catalyzed and noncatalyzed reaction, in the enzyme-catalyzed reaction the hydrogenation product is formed in a constraint conformation which relaxes upon dissociation from the enzyme. This exergonic conformational change could help to avoid product inhibition of the enzyme.