A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

Synthesis of [75Se]trimethylselenonium iodide from [75Se]selenocystine

N-Acetylglucosamine-6-sulfate sulfatase activity was assayed by incubation of the radiolabeled monosaccharide N-acetylglucosamine [1-14C]6-sulfate (GlcNAc6S) with homogenates of leukocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with N-acetylglucosamine-6-sulfate sulfatase deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls and other mucopolysaccharidosis types. The level of enzymatic activity toward GlcNAc6S was compared with that toward a sulfated disaccharide and a sulfated trisaccharide prepared from heparin. The disaccharide was desulfated at the same rate as the monosaccharide and the trisaccharide at 30 times that of the monosaccharide. Sulfatase activity toward glucose 6-sulfate and N-acetylmannosamine 6-sulfate was not detected. Sulfatase activity in fibroblast homogenates with GlcNAc6S exhibited a pH optimum at pH 6.5, an apparent Km of 330 mumol/liter, and inhibition by both sulfate and phosphate ions. The use of radiolabeled GlcNAc6S substrate for the assay of N-acetylglucosamine-6-sulfate sulfatase in leukocytes and skin fibroblasts for the routine enzymatic detection of the Sanfilippo D syndrome is recommended.     

Uptake of75Se in cataractous rat lens proteins

The purpose of the present study was to measure the pattern of uptake of⁷⁵Se into proteins in normal rat lenses and into the proteins of lenses with selenite-induced cataract. Ten-day-old suckling rats received a single injection of⁷⁵Se with or without a cataractous dose of cold carrier sodium selenite. Four days after injection, the proteins from excised lenses were counted for⁷⁵Se radioactivity and subjected to gel permeation chromatography, amino acid analyses, and mass spectrometry. All three soluble crystallin lens proteins took up⁷⁵Se in both normal and cataractous lenses. However, cataractous lenses did not take up⁷⁵Se into a soluble protein in which major quantities of⁷⁵Se were taken up in normal rats. Futhermore,⁷⁵Se in the gamma-crystallins was associated with an unusual acidic amino acid. It was concluded that selenium metabolism by lens proteins may be unusual compared to other soft tissues.     

Effect of lead on the intestinal absorption of sodium selenite and selenomethionine (75Se) in chicks

The present study was designed to investigate the interactions of lead with the intestinal absorption of selenium in chicks. The absorption of⁷⁵Se-selenite and⁷⁵Se-methionine was determined in 3-wk-old white Leghorn cockerels using both thein situ ligated intestinal loop procedure and oral administration of the isotopes. The highest lead concentration (1 mM) significantly reduced the percentage of⁷⁵Se-selenite absorbed from thein situ ligated duodenal loop, but did not influence the retention of the orally administered isotope. Neither was the absorption of⁷⁵Se-methionine affected by the presence of Pb in the intraduodenal dosing solution. The above experiments suggest that the direct luminal interaction of lead with the absorption of selenium is not of practical importance. On the other hand, feeding 1000 ppm Pb in the diet prior to the measurement of selenium absorption significantly reduced the transfer of the radioactive selenite from the intestinal lumen to the body. The mechanism of this effect involved enhanced retention of the⁷⁵Se-selenite by the intestinal tissue. The inhibitory effect of lead exposure on selenium absorption also appeared to be alleviated by the increase in the dietary selenium content.     

A conjugate counting method to determine [75Se]SeHCAT retention in the human body

To evaluate the functional integrity of the distal part of the ileum the retention of a γ-labelled bile acid (SeHCAT) in the human body can be measured with a detector. Due to the lack of a whole body counter at our institution a two detector system was designed to measure SeHCAT retention and an evaluation of such a system has been made. The detectors are positioned on either side of a patient lying supine on a hospital trolley. The trolley is stepped forward in 100 mm steps, to determine the SeHCAT activity in the patient. With these counts the location of the SeHCAT activity and total activity present in the body can be determined. A water rilled phantom and a phantom consisting of nine 1-L saline bags with ⁷⁵Se activity placed in them was used to determine system performance. Four patients with no history of bowel disease were compared with published data for normals. Results showed that the system performed satisfactorily, and accurate quantitative measurements could be made, showing that this inexpensive system could be used where a whole body counter is not available.     

Uptake of selenium-75 by PHA-stimulated lymphocytes

To determine which of a variety of inorganic and organic selenium compounds could best stimulate glutathione peroxidase, human lymphocytes were cultured with a number of selenium sources. The phytohemagglutinin-transformed lymphocytes were cultured in the presence of⁷⁵Se bound to serum proteins (25% v/v) or 10^?7 M concentrations of [⁷⁵Se]-selenite, [⁷⁵Se]-selenate, [⁷⁵Se]-selenocystine, and [⁷⁵Se]-selenomethionine. Organic forms of selenium were taken up in preference to inorganic forms. Control cultures, from which exogenous selenium had been omitted, showed a decreased level of glutathione peroxidase activity at the end of a 4 d culture period. Of the Se sources tested, [⁷⁵Se]-selenocystine and [⁷⁵Se]-labeled fetal calf serum proteins increased enzyme activity significantly, 79 and 47%, respectively, but selenite increased activity only by 7%. These results indicate that selenium from the two organic sources is most readily available for glutathione peroxidase synthesis.     

Use of a simple gamma flow cell to monitor chromatography column effluents containing 75Se.

Scintillation flow cells provide a convenient means of monitoring column effluents for radioactively labeled compounds. The use of flow cells to monitor effluents containing ³H, ¹⁴C, and other β-emitting isotopes is well established (1) and such cells are readily available as accessories for use with liquid scintillation counters. β-Flow cells have occasionally been used to monitor γ emitters [e.g., Martin et al. (2)] but the counting efficiency is extremely low even for weak γ emitters such as ⁷⁵Se, due to the limited mass of scintillant available to absorb the radiation.Spencer et al. (3) described a simple γ flow cell consisting of a coiled polyethylene tube inserted in to the well crystal of a γ counter, but no information on counting efficiency was given. Although spiral plastic γ cells have been manufactured commercially (e.g., Nuclear Enterprises Ltd., NE702¹) they are not offered as a standard accessory for most common makes of γ counter and do not appear to be readily available from commercial sources.This communication describes a simple, low cost γ flow cell constructed for use in a study on selenium transport in higher plants, which involved the separation of ⁷⁵Se-labeled constituents in a large number of samples of xylem sap.     

Selenium in the anterior pituitary of the rat after a single injection of75Se sodium selenite

In order to investigate the selenite metabolism in the anterior pituitary and compare it with other endocrine organs, rats were injected intraperitoneally with⁷⁵Se sodium selenite (5 mg/kg). The rats were whole body counted shortly after injection and recounted just before sacrifice, which was performed 2, 24, 48 h, and 4, 10, 20, 30, 40, 60, and 80 d after injection. Besides the anterior pituitary, the selenium content was also estimated in the thyroid gland, testis, adrenals, liver, kidney, and blood. The maximum selenium content was observed in all organs 2 h after injection, at which time the anterior pituitary contained 2.9 μg/g wet wt, compared to 13.5 μ/g wet wt in liver and .6 μg/mg wet wt in testis. The excretion of selenite from the anterior pituitary resembled that seen in most other organs investigated, i.e., an initial rapid excretion and a slower secondary phase resembling a first order reaction. Practically all selenium was excreted by 60 d after injection.     

Ab initio study of singlet excited states of bicyclo[2.2.0]hexasilane and tricyclo[4.2.0.02,5]octasilane

The singlet excited states in the optimized geometries of bicyclo[2.2.0]hexasilane and tricyclo[4.2.0.0^2,5]octasilane are studied theoretically using the ab initio molecular orbital method at the RHF/3-21G^* level with the all-singles CI approximation. These molecules are found to have zigzag structures in both the ground states and the singlet excited states corresponding to the lowest absorptions, and the estimated Stokes shifts between the lowest absorption and fluorescence are similar to the experimental values.     

Metabolism of N6,O2'-[3H]dibutyryl cyclic adenosine 3',5'-monophosphate and macromolecular interactions of the products perfused rat liver.

Mitochondria from liver, kidney, brain, and skeletal muscle metabolized acetaldehyde. Acetaldehyde oxidation by liver and kidney mitochondria was maximal at low levels of acetaldehyde and was sensitive to rotenone, suggesting the involvement of a NAD⁺-dependent aldehyde dehydrogenase with a high affinity for acetaldehyde. Acetaldehyde oxidation was stimulated 50% by ADP, suggesting that, in state 4, reoxidation of NADH is rate limiting for acetaldehyde oxidation. In state 4, acetaldehyde oxidation was decreased by NAD⁺-dependent substrates, as well as by succinate and ascorbate. The inhibition by the latter two substrates was prevented by ADP, dinitrophenol, valinomycin, and gramicidin, but not by oligomycin. Since these compounds are linked to energy transduction and utilization, the data suggest that the inhibition is mediated via energy-dependent reversed electron transport. In state 3, all of these substrates caused considerably less inhibition of acetaldehyde oxidation, suggesting that the activity of aldehyde dehydrogenase, and not of NADH reoxidation, is probably rate limiting for acetaldehyde oxidation. The ionophores valinomycin and gramicidin stimulated acetaldehyde oxidation to a greater extent than ADP. These ionophores also stimulated acetaldehyde oxidation in the presence of ADP. Stimulation by valinomycin occurred in the presence of monovalent cations transported by this ionophore, e.g., K⁺, Rb⁺, Cs⁺. Stimulation by gramicidin also occurred in the presence of these cations, but did not occur with Na⁺ or Li⁺. Na⁺ prevents the stimulation of acetaldehyde oxidation, which occurs in the presence of gramicidin and K⁺. The stimulation by valinomycin and gramicidin was energy dependent and required the presence of a permeant anion. In the absence of an ionophore, potassium phosphate had no effect on acetaldehyde oxidation. These data suggest that the oxidation of acetaldehyde by rat liver and kidney mitochondria is influenced by the oxidation-reduction state of the mitochondria and by the cationic environment. With brain and muscle mitochondria, the rate of acetaldehyde oxidation increased two- to threefold as the concentration of acetaldehyde was raised from 0.167 to 0.50 mm. Acetaldehyde oxidation in these mitochondria was also sensitive; to rotenone, indicating dependence on NAD⁺. ADP, valinomycin, gramicidin, and succinate, compounds which either increased or decreased the rate of acetaldehyde oxidation by liver and kidney mitochondria, had no effect on acetaldehyde oxidation by muscle or brain mitochondria. In state 4, mitochondria from Becker-transplantable hepatocellular carcinoma HC-252 oxidized acetaldehyde at the same rate as liver mitochondria. However, in the presence of ADP, dinitrophenol, valinomycin and gramicidin, the rate of acetaldehyde oxidation by the tumor mitochondria was two to three times greater than that of liver mitochondria, suggesting the presence of a more active; acetaldehyde-oxidizing system in tumor than in liver mitochondria.     

Fluctuations in Fe,Cu, Zn,Br, As,Se, and Rb concentrations in C57L/J mice bearing BW7756 murine hepatoma using radioisotope-induced X-ray fluorescence

The effects of selenite or selenate supplementation on the concentration and distribution of Fe, Cu, Zn, As, Br, and Rb are investigated using the radioisotope-induced X-ray fluorescence, RIXRF. These effects are studied in the animals bearing BW7756 murine hepatoma and healthy animals for both of the oxidation states. Selenite and selenate induce different effects on the distribution of selenium, zinc, copper, bromine, and rubidium. The differences may be attributed to the differences in the inter element interaction after absorption into the bloodstream as well as to the mode of their intestinal absorption. Simultaneous supplementation of copper with selenite or selenate at the described levels has a profound influence on the concentration levels of other elements in the normal as well as in the diseased mice. The administration of selenium (0.67 μg/g body wt sodium selenite or sodium selenate, daily) and selenium and copper (0.67 and 1.35 μ/g body wt, respectively) has no effect on the incidence rate of hepatoma development.     

Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate

Two methods were investigated for the no-carrier-added synthesis of N-succinimidyl 4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). The first, an attempted nucleophilic aromatic substitution by [¹⁸F]fluoride on N-succinimidyl 4-nitrobenzoate was unsuccessful. The second method involved three steps; [¹⁸F]fluoride for trimethylammonium substitution on 4-formyl-N,N,N-trimethylanilinium triflate, oxidation to 4-[¹⁸F]fluorobenzoic acid, followed by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide to form S[¹⁸F]FB. Total synthesis and purification time was 100 min and the overall radiochemical yield was 25% (decay corrected). A monoclonal antibody F(ab′)₂ fragment could be labeled in 40–60% yield by reaction with S[¹⁸F]FB for 15–20 min. The tissue distribution in normal mice and in vitro tumor binding of the antibody F(ab′)₂ labeled by reaction with S[¹⁸F]FB were comparable to those observed for the fragment after radioiodination using N-succinimidyl 4-[¹²⁵I]iodobenzoate.     

Tumor uptake studies of S-adenosyl-l-[methyl-11C]methionine and l-[methyl-11C]methionine

Tumor accumulation of S-adenosyl-l-[methyl-¹¹C]methionine ([¹¹C]SAM) was investigated in mice bearing mammary carcinoma (FM3A) and in rats bearing ascitic hepatoma (AH109A). After injection of [¹¹C]SAM the blood clearance of ¹¹C radioactivity was rapid. The ¹¹C level was relatively high in both tumors. The uptake ratios of tumor to organ increased with time in several organs, especially in brain and muscle. In FM3A tumor tissue the ¹¹C was incorporated with time into the acid-precipitable fraction and 38% of the ¹¹C was detected in this fraction at 60 min after injection. This fraction reflects the amount of ¹¹C-methyl group transferred into macromolecules in tumor tissue. In AH109A-bearing rats the metabolisms of [¹¹C]SAM and l-[methyl-¹¹C]methionine ([¹¹C]Met), in vivo precursor of SAM, were compared. Tumor uptake of [¹¹C]SAM was about two thirds of that of [¹¹C]Met at 20 min after injection. At this time, for the [¹¹C]SAM 27 and 8% of the ¹¹C in the AH109A tissue were detected in the acid-precipitable and the lipid fractions, respectively. The corresponding figures for [¹¹C]Met were 61% and 2%. In the liver considerable amounts of ¹¹C were observed in the lipid fraction for both tracers.These results show that [¹¹C]SAM has potential as a tracer for tumor localization with positron emission tomography (PET) and suggest that in tumor studies combining [¹¹C]Met and PET, it should be taken into account that the ¹¹C-labeled methyl group of [¹¹C]Met is not only incorporated into protein but also other macromolecules and lipids via [¹¹C]SAM.     

Differential protein expression due to Se deficiency and Se toxicity in rat liver

There is a U-shaped dose-response between selenium (Se) status and health outcomes, but underlying metabolic processes are unclear. This study aims to identify candidate proteins in liver regulated by dietary Se, ranging from deficiency to toxic. Male rats (n=4) were fed graded Se concentrations as selenite for 28 days. Bulk Se analysis was performed by ICP-MS on both soluble and insoluble fractions. Soluble fraction samples were chromatographically separated for identification of selenocompounds by SEC-ICP-MS and protein quantification by LC-MS/MS. Bioinformatics analysis compared low-Se (0 and 0.08 µg Se g⁻¹) and high-Se (0.8, 2 and 5 µg Se g⁻¹) with adequate-Se (0.24 µg Se g⁻¹) diets. Major breakpoints for Se were seen at 0.8 and 2 µg Se g⁻¹ in the insoluble and soluble fractions, respectively. Glutathione peroxidase 1 protein abundance reached a plateau at ≥0.08 µg Se g⁻¹diet; Se bound to selenium binding protein 2 was observed with 2 and 5 µg Se g⁻¹ Se. The extreme diets presented the highest number of differentially expressed (P value <0.05, FC ≥1.2) proteins in comparison to the adequate-Se diet (0 µg Se g⁻¹: 45 proteins; 5 µg Se g⁻¹: 59 proteins); 13 proteins were commonly affected in 0 and 5 µg Se g⁻¹ treatments. Network analysis revealed that the metabolism of glutathione, xenobiotics and amino acids were enriched in both 0 and 5 µg Se g⁻¹ diets, indicating a U-shape effect of Se. This similarity is likely due to down-stream effects of lack of essential selenoproteins in Se deficiency and due to toxic effects of Se that exceeds the capacity to cope with excess Se.     

Significance of Bacterial Activity for the Distribution and Dynamics of Transparent Exopolymer Particles in the Mediterranean Sea

The study of transparent exopolymer particles (TEP) in the Mediterranean Sea is particularly relevant as they can be promoters of mucilage events, a frequent phenomenon there. We assessed the influence of bacterioplankton on TEP distribution and dynamics across the west–east axis of the Mediterranean Sea. We performed an extensive study of TEP, dissolved carbohydrates, and their relationships with bacterial abundance and bacterial production (BP). A significant and positive relationship was observed between BP and TEP in the study region (r ²?=?0.51, p?<?0.001). The direct release of TEP by bacteria was experimentally corroborated using regrowth cultures where increases in TEP tracked bacterial growth in abundance and production. These TEP increases were positively related to the increases in BP (r ²?=?0.78, p?<?0.05). The consistency (similar slopes) of the regression lines between BP and TEP in natural conditions and between the increases of BP and TEP in the experiments underlines the relevant role of bacteria in the formation of TEP in this area.     

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD⁺]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD⁺]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD⁺]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD⁺]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD⁺]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.     

Hypoxia Imaging Using PET and SPECT: The Effects of Anesthetic and Carrier Gas on [64Cu]-ATSM, [99mTc]-HL91 and [18F]-FMISO Tumor Hypoxia Accumulation

Background

Preclinical imaging requires anaesthesia to reduce motion-related artefacts. For direct translational relevance, anaesthesia must not significantly alter experimental outcome. This study reports on the effects of both anaesthetic and carrier gas upon the uptake of [⁶⁴Cu]-CuATSM, [^99mTc]-HL91 and [¹⁸F]-FMISO in a preclinical model of tumor hypoxia.

Methodology/Principal Findings

The effect of carrier gas and anaesthetic was studied in 6 groups of CaNT-bearing CBA mice using [⁶⁴Cu]-CuATSM, [^99mTc]-HL91 or [¹⁸F]-FMISO. Mice were anaesthetised with isoflurane in air, isoflurane in pure oxygen, with ketamine/xylazine or hypnorm/hypnovel whilst breathing air, or in the awake state whilst breathing air or pure oxygen. PET or SPECT imaging was performed after which the mice were killed for organ/tumor tracer quantitation. Tumor hypoxia was confirmed. Arterial blood gas analysis was performed for the different anaesthetic regimes. The results demonstrate marked influences on tumor uptake of both carrier gas and anaesthetic, and show differences between [^99mTc]-HL91, [¹⁸F]-FMISO and [⁶⁴Cu]-CuATSM. [^99mTc]-HL91 tumor uptake was only altered significantly by administration of 100% oxygen. The latter was not the case for [¹⁸F]-FMISO and [⁶⁴Cu]-CuATSM. Tumor-to-muscle ratio (TMR) for both compounds was reduced significantly when either oxygen or anaesthetics (isoflurane in air, ketamine/xylazine or hypnorm/hypnovel) were introduced. For [¹⁸F]-FMISO no further decrease was measured when both isoflurane and oxygen were administered, [⁶⁴Cu]-CuATSM did show an additional significant decrease in TMR. When using the same anaesthetic regimes, the extent of TMR reduction was less pronounced for [⁶⁴Cu]-CuATSM than for [¹⁸F]-FMISO (40–60% versus 70% reduction as compared to awake animals breathing air).

Conclusions/Significance

The use of anaesthesia can have profound effects on the experimental outcome. More importantly, all tested anaesthetics reduced tumor-hypoxia uptake. Anaesthesia cannot be avoided in preclinical studies but great care has to be taken in preclinical models of hypoxia as anaesthesia effects cannot be generalised across applications, nor disease states.     

Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.     

77Se Enrichment of Proteins Expands the Biological NMR Toolbox

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable ⁷⁷Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate ⁷⁷Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, ⁷⁷Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.