The catalysis of superoxide dismutation ( 2 0 1 + 2H+ -H202 + 0,) by manganese superoxide dismutase (MnSOD) from Thermus Thermophilus was examined by stopped-flow spectrophotometry. As found earlier by McAdam et al. [McAdam, M. E.; Fox, R. A.; Lavelle, F.; Fielden, E. M. Biochem. J . 1977, 165, 81-87], decay curves of 01 in the presence of MnSOD from Bacillus Stearothermophilus are characterized by three distinct phases: rapid disappearance of 02-(the "burst" phase), a period of approximately zero-order disappearance of 02-(the "steady-state" phase), and a very rapid depletion of 02-toward the end of the reaction. The enzyme from T. Thermophilus shows a similar kinetic pattern, and our data provide a chemical explanation for this behavior: The molar consumption of 0; in the burst phase is ([O2-Ie/[MnlT) -80. The magnitude of the burst is decreased -2.5-fold in D20, whereas the zero-order phase is the same in both solvents. This indicates that proton transfer is probably the rate-limiting step when the enzyme is saturated with 0; and that the reaction by which inactive enzyme returns to active enzyme is not limited by proton transfer. At low temperatures (2-6 "C) in D20, the overall reaction was sufficiently slow to allow observation of spectral changes associated with the metal chromophore during the steady state, and we were able to obtain an absorption spectrum of the enzyme during this period. This was assigned to the inactive form of the enzyme and is characterized by a band near 650 nm (c -230 [Mnl-' cm-I) and a band near 410 nm (c -700 [Mnl-' cm-I). We speculate that inactivation of the enzyme occurs by oxidative addition of 0; to Mn(II), within a Michaelis complex, forming a cyclic peroxo complex of Mn(II1) with the reverse of this reaction yielding active enzyme. k j -650 S-' Mn":02-+ Mn"':o22k-5 -10 s-' A reaction scheme composed of a cyclic redox process, as described previously for the FeSOD of Escherichia coli [Bull, C . ; Fee, J. A. J . Am. Chem. SOC. 1985, 107, 3295-33041, and the above reversible side reaction adequately account for the kinetic behavior of MnSODs.In previous communications we described the steady-state kinetic properties of the iron-containing superoxide dismutase from E. coli,' of Cu/Zn-containing superoxide dismutase (SOD35) from bovine tissues,* and of FeEDTA.3v35 In this paper we describe our studies with the manganese-containing protein from Thermus thermophilus.There are three types of superoxide dismutases as determined by the metal involved in the catalysis of reaction 1: Cu, Fe, and Mn. The general properties, distribution, and possible biological (1) function of these proteins have been discussed in a large number of review and discussion papers (cf. Ref 1 of ref 1 for reviews). The present work is concerned with manganese-containing superoxide dismutases, and the object of study is the protein from Thermus thermophilus. This protein is a tetramer of 21 kDa subunits, each of which binds, on the average, -0.6 Mn(II1) ions. Solutions of the protein have a reddish-pur...

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Leukocytes comprise less than 1% of all blood cells. Enrichment of their number, starting from a sample of whole blood, is the required first step of many clinical and basic research assays. We created a microfluidic device that takes advantage of the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, to separate leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels designed to enhance lateral migration of leukocytes and their subsequent extraction from the erythrocytedepleted region near the sidewalls. A single pass through the device produces a 34-fold enrichment of the leukocyte-to-erythrocyte ratio. It operates on microliter samples of whole blood, provides positive, continuous flow selection of leukocytes, and requires neither preliminary labeling of cells nor input of energy (except for a small pressure gradient to support the flow of blood). This effortless, efficient, and inexpensive technology can be used as a lab-on-a-chip component for initial whole blood sample preparation. Its integration into microanalytical devices that require leukocyte enrichment will enable accelerated transition of these devices into the field for point-of-care clinical testing.The rapidly expanding field of lab-on-a-chip microanalytical devices promises inexpensive, portable, miniaturized tools that could potentially revolutionize clinical and basic research analyses by dramatically reducing sample size and handling.1 -3 An important application of this technology is the analysis of leukocytes (white blood cells) or their contents, for which these cells must first be isolated from the whole blood sample. [4][5][6][7] Blood is a 45% suspension of cells (erythrocytes, platelets, leukocytes) in plasma. Erythrocytes (red blood cells) constitute the vast majority of all blood cells and are normally ∼1000 times more abundant than leukocytes. Traditionally, several milliliters of whole blood are drawn and then centrifuged in order to separate blood cells of different density. Plasma, platelets, and white and red cells can be separated by this procedure, but it is a labor-, energy-, and time-intensive process that relies on special equipment and requires trained personnel. 8, 9 Many current microanalytical devices have no integrated sample