Superoxide dismutase (SOD) is an enzyme that detoxifies superoxide (O2.-), a potentially toxic oxygen-derived species. Attempts to increase intracellular concentrations of SOD by direct application are complicated because SOD, being a relatively large molecule, does not readily cross cell membranes. We have identified a set of stable nitroxides that possess SOD-like activity, have the advantage of being low molecular weight, membrane permeable, and metal independent, and at pH 7.0 have reaction rate constants with O2.- ranging from 1.1 x 10(3) to 1.3 x 10(6) M-1 s-1. These SOD mimics protect mammalian cells from damage induced by hypoxanthine/xanthine oxidase and H2O2, although they exhibit no catalase-like activity. In addition, the nitroxide SOD mimics rapidly oxidize DNA-FeII and thus may interrupt the Fenton reaction and prevent formation of deleterious OH radicals and/or higher oxidation states of metal ions. Whether by SOD-like activity and/or interception of an electron from redox-active metal ions they protect cells from oxidative stress and may have use in basic and applied biological studies.

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Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O(2) and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 microM(-1) s(-1)) and dissociation (<0.006 s(-1)) of O(2) are among the slowest known, precluding transport or diffusion of O(2) as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25- and 77-fold increases in the combination rate constants for O(2) and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the chi((2,1)) dihedral angle for the indole ring increases progressively from approximately 93 degrees to at least 100 degrees in going sequentially from the deoxy to CO to O(2) derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O(2) dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.

Resistive-pulse sensing with biological or solid-state nanopores and nanopipettes has been widely employed in detecting single molecules and nanoparticles. The analytical signal in such experiments is the change in ionic current caused by the molecule/particle translocation through the pipet orifice. This paper describes a new version of the resistive-pulse technique based on the use of carbon nanopipettes (CNP). The measured current is produced by electrochemical oxidation/reduction of redox molecules at the carbon surface and responds to the particle translocation. In addition to counting single entities, this technique enables qualitative and quantitative analysis of the electroactive material they contain. Using liposomes as a model system, we demonstrate the capacity of CNPs for (1) conventional resistive-pulse sensing of single liposomes, (2) electrochemical resistive-pulse sensing, and (3) electrochemical identification and quantitation of redox species (e.g., ferrocyanide, dopamine, and nitrite) contained in a single liposome. The small physical size of a CNP suggests the possibility of single-entity measurements in biological systems.