The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 °C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1= 1.4 × 107 M−1 s−1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+· FeIVO). Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2= 65 s−1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome‐c peroxidase, was assigned to a two‐electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+· which was formed by an intramolecular electron transfer from tyrosine to the porphyrin‐π‐cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.

Purified prostaglandin H synthase (EC 1.14.994, reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with 02, as well as prostaglandin G2 or H 2 0 2 , decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12°C. From the conditions for the formation and the effect of inhibitors,

An optical real-time affinity biosensor, which is based on a monolithic silicon optoelectronic transducer and a microfluidic module, is described. The transducer monolithically integrates silicon avalanche diodes as light sources, silicon nitride optical fibers, and p/n junction detectors and efficiently intercouples these elements through a self-alignment technique. The transducer surface is hydrophilized by oxygen plasma treatment, silanized with (3-aminopropyl)triethoxysilane and bioactivated through adsorption of the biomolecular probes. The use of a microfluidic module allows real-time monitoring of the binding reaction of the gold nanoparticle-labeled analytes with the immobilized probes. Their binding within the evanescent field at the surface of the optical fiber causes attenuated total reflection of the waveguided modes and reduction of the detector photocurrent. The biotin-streptavidin model assay was used for the evaluation of the analytical potentials of the device developed. Detection limits of 3.8 and 13 pM in terms of gold nanoparticle-labeled streptavidin were achieved for continuous- and stopped-flow assay modes, respectively. The detection sensitivity was improved by silver plating of the immobilized gold nanoparticles, and a detection limit of 20 fM was obtained after 20-min of silver plating. In addition, two different analytes, streptavidin and anti-mouse IgG, were simultaneously assayed on the same chip demonstrating the multianalyte potential of the sensor developed.

Free radicals have been postulated to play an important role as mediators in the pathogenesis of shock syndrome and multiple-organ failure. We attempted to directly detect the increased formation of radicals by Electron Spin Resonance (ESR) in animal models of shock, namely the endotoxin (ETX) shock or the hemorrhagic shock of the rat. In freeze-clamped lung tissue, a small but significant increase of a free radical signal was detected after ETX application. In the blood of rats under ETX shock, a significant ESR signal with a triplet hyperfine structure was observed. The latter ESR signal evolved within several hours after the application of ETX and was localized in the red blood cells. This signal was assigned to a nitric oxide (NO) adduct of hemoglobin with the tentative structure [alpha 2+ NO)beta 3+)2. The amount of hemoglobin-NO formed, up to 0.8% of total hemoglobin, indicated that under ETX shock a considerable amount of NO was produced in the vascular system. This NO production was strongly inhibited by the arginine analog NG-monomethyl-arginine (NMMA). The ESR signal of Hb-NO was also observed after severe hemorrhagic shock. There are three questions, namely (i) the type of vascular cells and the regulation of the process forming such a large amount of NO during ETX shock, (ii) the pathophysiological implications of the formed NO, effects which have been described as cytotoxic mediator, endothelium-derived relaxing factor (EDRF) or inhibitor of platelet aggregation, and (iii) the possible use of Hb-NO for monitoring phases of shock syndrome.