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Two new species of dwarfgobies are described, Eviota asymbasia from the Sulu Sea, Philippine Islands, south to Java and west to Damar, and E. bipunctata occurring from the Indian Ocean eastward into the western Pacific Ocean. Eviota asymbasia lacks the IT pore of the cephalic sensory-pore system and usually also the POP, has a dorsal/anal-fin formula of 8/8, has some pectoral-fin rays branched and usually 15 rays, two spots on the pectoral-fin base, no dark spot over the preural centrum, first dorsal fin may be filamentous, six ventral postanal spots, and the male genital papilla is not fimbriate. Eviota bipunctata belongs to the group with cephalic sensory-pore system pattern 2 (lacking only the IT pore), some pectoral-fin rays branched, pectoral-fin base with 1 or 2 prominent dark spots, dorsal/anal-fin formula usually 8/8, and no occipital spots.

The "peroxidase" activity of the copper-zinc superoxide dismutase is a poorly sustained activity because of the competing inactivation of the enzyme. New evidence suggests that the bound oxidant may be partitioning between oxidizing the enzyme or oxidizing small anions. At constant peroxide, nitrite and azide only partially protect the enzyme (50%) against loss of copper(I) and inactivation up to one anion per copper. Beyond that level, there is no further protection. Bicarbonate ion also protects, but larger amounts are required. These data suggest that there is significant oxidation of the enzyme even in the presence of the small anions and therefore the formation of the bound oxidant cannot be sustained in a true catalytic process., (Copyright 2000 Academic Press.)

This work summarizes observations from numerous investigators on the reaction of the copper-zinc superoxide dismutase with hydrogen peroxide at physiological pH in order to propose a likely sequence of events that leads to 2-oxo-histidine formation, copper loss, inactivation, and random and site-specific peptide fragmentation. New data is presented for the bovine liver enzyme that indicate copper is lost as the copper(I) form which immediately reacts with bathocuproine disulfonate to form the characteristic complex that absorbs at 485 nm. Studies in TRIS buffer ruled out the loss of copper(II) followed by reduction of the high potential copper(II)-bathocuproine disulfonate complex by buffer because TRIS is known not to reduce this complex. The rate of loss of copper(I) is not affected by the spin trap, 5,5'-dimethylpyrolline-N-oxide (DMPO), nor by replacing oxygen with argon in the reaction. In addition, changes in the native electrophoretic pattern that are correlated with copper loss and not peptide fragmentation are also unaffected by DMPO, argon, EDTA, or DTPA. These data are taken as indirect evidence that the formation of 2-oxo-histidine is the first oxidative event, unaffected by DMPO, that occurs at the bound oxidant and leads to loss of copper(I). Peptide fragmentation and the peroxidative activity of the dismutase are discussed in light of these observations.

N,N'-Diethyldithiocarbamate has been shown to be an analytical stain for copper in native polyacrylamide gels of the copper-zinc superoxide dismutase from purified preparations as well as from crude red cell extracts, i.e., lysates from which hemoglobin has been removed (Jewett, S. L., and Rocklin, A. M. (1994) Anal. Biochem. 217, 236-240). Applying this methodology, it was found that the relative amounts of copper-containing forms of copper-zinc superoxide dismutase (EC 1.15.1.1) from bovine red cell extracts did not change significantly with either the age or with hydrogen peroxide treatment of the red cells. Furthermore, no significant changes were seen in the specific activity of the dismutase in either type of experiment. These observations for both types of experiments are contrary to what was expected from similar studies reported in the literature. However, discrepancies may be accounted for by hemoglobin interference in indirect dismutase assays of the previous work. In the case of the peroxide treatment of red cells, however, there is an additional factor in that the dismutase is protected from peroxide-mediated changes in copper content and heterogeneity by the hemoglobin present. This protection was demonstrated in in vitro experiments using only a 24-fold excess of hemoglobin over the dismutase.

N,N'-Diethyldithiocarbamate reacts with copper in the copper-zinc superoxide dismutase (EC 1.15.1.1) in polyacrylamide gels to form stable yellow-brown bands that are quantifiable at 448 nm. This method of examining superoxide dismutase has been applied to crude extracts of the enzyme obtained from red cell lysates from which hemoglobin has been removed by chloroform-ethanol precipitation. This treatment did not affect the activity and heterogeneity of purified dismutase added to lysates and recovered by the same method. The bands that develop in the dithiocarbamate-stained gels of the extracts correspond exactly to the bands of dismutase activity obtained with a positive activity stain using dianisidine, indicating that the dismutase is the only copper protein that gives rise to these bands. The amount of superoxide dismutase in the bands, determined by comparing the areas under unknown peaks to areas obtained with a standard dismutase sample, agrees with the amount predicted from indirect superoxide dismutase activity measurements. Any color in gels due to trace hemoglobin or hemoglobin degradation products is bleached overnight during staining with the dithiocarbamate.

Although numerous reviews and articles have been written about indirect superoxide dismutase assays, little has been reported about the variation of the amount of superoxide dismutase giving 50% inhibition (SOD50), defined as one unit of dismutase activity, with variations in assay conditions. This report indicates that there is a linear dependence of the SOD50 on the magnitude of the uninhibited rate of oxidation of the organic substance for two indirect assays, one involving epinephrine autoxidation and the other NADH oxidation. For one indirect assay involving an acceleration of the oxidation rate of an organic substrate, the amount of superoxide dismutase giving a doubling of the oxidation rate, defined as one unit of dismutase activity, also shows this linear dependence on the autoxidation rate of the organic substance. The significance of this dependence and its implications for experimenters using these assays is discussed.

The autoxidation of catecholamines has been proposed to be a source of oxygen radicals in ischemia-reperfusion injury. However, this autoxidation per se is extremely slow at physiological pH and therefore is unlikely to be a primary source of oxygen radicals in ischemia-reperfusion injury. On the other hand, oxygen radicals from catecholamines are more likely to arise through catalyzed oxidations involving enzymatic systems and/or metal ions. It is these latter reactions that may be of interest with respect to damage associated with ischemia-reperfusion injury.

The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.