Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry, and "logic gate"-like dual functionality.

1Cellobiose dehydrogenase is a hemoflavoenzyme that catalyzes the sequential electron-transfer from an electron-donating substrate (e.g. cellobiose) to a flavin center, then to an electron-accepting substrate (e.g. quinone) either directly or via a heme center after an internal electron-transfer from the flavin to heme. We cloned the dehydrogenase from Humicola insolens, which encodes a protein of 761 amino acid residues containing an N-terminal heme domain and a C-terminal flavin domain, and studied how the catalyzed electron transfers are regulated. Based on the correlation between the rate and redox potential, we demonstrated that with a reduced flavin center, the enzyme, as a reductase, could export electron from its heme center by a "outer-sphere" mechanism. With the "resting" flavin center, however, the enzyme could have a peroxidase-like function and import electron to its heme center after a peroxidative activation. The dual functionality of its heme center makes the enzyme a molecular "logic gate", in which the electron flow through the heme center can be switched in direction by the redox state of the coupled flavin center.

A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale.

A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (< or = 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O2 or other acceptors. When D-glucose served as electron-donating substrate, an activity of 2 s(-1) was observed at pH 5.5 and 23 degrees C. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications.

A simplified method for determining the P50 of blood.

A method to determine the P50 of whole blood is described using a modified American Optical reflectance oximeter, pump, and membrane tonometer, together with PO2, PCO2, and pH measurements in a standard blood gas machine. Determinations of P50 were made in 66 patients and normal subjects and in two situations where P50 was very low and very high. The results were compared to oxygen saturations calculated from measured oxygen content. The directly determined oxygen saturation agreed with the assumed saturation of 50 per cent in the oximeter within 0.5 per cent. The apparatus appears to be a simple and relatively inexpensive method to obtain P50 as long as blood carboxyhemoglobin or methemoglobin contents are not elevated.