Microanalysis of non-heme iron in animal tissues

The method recommended by the Iron Panel of the International Committee for the Standardization in Haematology for measurement of serum iron was adapted for measurement of non-heme iron in animal tissues. The method developed was designed specifically to facilitate measurement of non-heme iron using as little as 10 mg of tissue, in a final reaction volume of 60 microl. In this assay, tissue homogenates are treated with hydrochloric acid and trichloroacetic acid and heated at 95 degrees C. Non-heme iron is released and protein is precipitated. Following centrifugation, iron in the supernatant is reacted with ferrozine in the presence of the reducing agent thioglycolic acid, and the complex is quantified by spectrophotometry. The method was validated by analysis of two Standard Reference Materials (bovine liver), comparing results of this assay against certified values and concentrations determined by flame atomic absorption spectrometry following acid digestion. Results using this method for analysis of non-heme iron in guinea pig tissues (liver, kidney and heart) compared favorably with those obtained using micro-scale adaptations of three published reference methods. The new method was more sensitive, required less time, and was less cumbersome than the three published methods to which it was compared.     

Non-ferritin, non-heme iron pools in rat tissues

Concentrations of intracellular, low molecular weight (LMW) and desferrioxamine B (DF) chelatable Fe, in tissues of normal, Fe-deficient and Fe-loaded female rats, were determined. Ice cold, high speed supernatants were rapidly fractionated on Ultrogel AcA202 or by filter centrifugation. After correction for residual blood and DF effects on Fe proteins, liver, kidney, heart and spleen contained 3-8 micrograms/g LMW Fe, brain 20 micrograms/g, with DF; two-thirds of this was detected without DF. There was little variation with Fe status. MW standardization and fractionation on Sephadex G-25 indicated components of apparent MW 13,000, 1400 and 350; the latter two were rapidly labeled with in vivo 59Fe.     

Modeling non-heme iron proteins

Synthetic modeling studies of non-heme iron proteins continue to contribute to our understanding of the mechanism of these proteins. Recently, mononuclear Fe(IV)=O complexes have been prepared and characterized to model the same species that are proposed to be the reactive intermediates in reactions involving mononuclear non-heme iron proteins. Generation of such species for the oxidation of organic substrates has also been demonstrated. Other advances include successful modeling of the structural and functional aspects of diiron non-heme proteins with the use of terphenyl-based carboxylate ligands and the development of several iron-based reagents that catalyze oxidation reactions with the use of various oxidants, including dioxygen.     

High-throughput fluorescence assay for membrane-protein interaction

Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

High-throughput screening assay for D-amino acid oxidase

A membrane-based high-throughput screening (HTS) assay for active d-amino acid oxidase (DAAO) in liquid samples as well as in intact Escherichia coli cells has been developed and optimized. The detection limit of the assay was less than 1 ng per sample. The method proposed can be used for quantitative DAAO determination in the range of 0.13 to 3.60 ng enzyme per probe. The protocol was successfully tested to screen a library of E. coli clones containing mutant DAAOs active toward target substrates.     

Magnetic circular dichroism of non-heme iron proteins

The magnetic circular dichroism (MCD) at 45 kgauss has been determined for a group of non-heme iron proteins. Both transferrin and conalbumin exhibit a single, positive ellipticity band at 330 nm ([θ]_M = 560). Oxy- and methemerythrin, spinach and clostridial ferredoxins and rubredoxin all display distinctive multibanded spectra which may reflect such factors as coordination of the metal, its ligands, metal bridging by other atoms, and varying degrees of metalmetal coupling. The MCD spectra of both ferredoxins and rubredoxin undergo dramatic change upon oxidoreduction providing a potential means for relating the electronic structure of the iron to protein function. In contrast to the plant ferredoxins, the magnetic field does not significantly affect the CD spectra of adrenodoxin and putidaredoxin.     

High-throughput scintillation proximity assay for transglutaminase activity measurement

Members of the transglutaminase enzyme family are involved in a broad range of biological phenomena, including haemostasis, apoptosis, semen coagulation, skin formation, and wound healing. A new and rapid method for measurement of transglutaminase activity is described in this article. The enzyme links tritium-labeled putrescine to biotinylated oligoglutamine, and the tritiated peptide is bound to a streptavidin-coated microtiter plate permanently covered by a thin layer of scintillant. Only the radioisotope incorporated into the peptide substrate is close enough to the scintillant molecules for photons to be produced. The signal generation depends on the transglutaminase activity, and it can be detected by appropriate light-measuring instrumentation without separation steps. The assay is sensitive, specific, linear at concentrations of tissue transglutaminase between 0.05 and 1.6m U/ml, and suitable for high-throughput measurements.     

4-Nitrocatechol as a colorimetric probe for non-heme iron dioxygenases.

4-Nitrocatechol is examined as an active site probe for non-heme iron dioxygenases and found to be of value, particularly with those containing iron in the Fe(II) oxidation state. 4-Nitrocatechol is astrong competitive inhibitor of substrate oxygenation by protocatechuate 3,4-dioxygenase, forming a reversible complex with this enzyme, and by pyrocatechase. The number of binding sites per enzyme molecule titrated spectrophotometrically with 4-nitrocatechol agrees with results from previous studies with either the principal substrate or other analogues, as expected of an effective probe. Despite these facts and the observation that both enzymes cleave the same substrates at the same carbon-carbon bond, the optical and electron paramagnetic resonance (EPR) spectra of their 4-nitrocatechol complexes are remarkably different. The 4-nitocatechol-protocatechuate 3,4-dioxygenase optical spectra resemble that of the 4-nitrocatecholate ion shifted 20 to 30 nm to longer wavelength. Concomitant with this change the EPR signal centered at g equal 4.28 shows increased rhombicity (g values at 4.74, 4.28, and 3.74).In contrast, the spectrum of the 4-nitrocatechol-pyrocatechase complex has a maximum at the same wavelength as that of a 1:1 solution of free Fe(II) and 4-nitrocatechol in the absence of enzyme after titration of the catecholic protons with base and the g equal 4.28 EPR signal is not resolved at liquid N-2 temperature. These changes are interpreted as resulting in part from a pronounced change in the ligand fields about the irons at the active sites which in the case of protocatechuate 3,4-dioxygenase leads to enzyme inactivation. The results also are the first indication that substrate analogues change their ionization form upon complexation with Fe (III) dioxygenases. The interaction of the probe with metapyrocatechase, an Fe(III) containing dioxygenase, and with several additional oxygenases and hydroperoxidases is also briefly examined. The probe is not specific for any particular class of non-heme iron dioxygenases.     

High-throughput solubility assay for purified recombinant protein immunogens

A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.     

High-throughput microtiter assay for Hoechst 33342 dye uptake

Exclusion of Hoechst 33342 dye is a characteristic common to stem cells, as well as chemotherapy-resistant cancer cells. Normally, these dye-excluding cells can be sorted from enzymatically dissociated tissues with a UV cell sorter/flow cytometer. UV-flow cytometry can be expensive, time-consuming and not readily available to all laboratories. We have developed a simple, high-throughput 96-well microtiter plate assay by which cell populations can be quickly screened for Hoechst dye uptake and exclusion. The method is compatible with green-fluorescent EGFP expressing cells, often used in stem cell biology. Useful applications for this assay will be the rapid screening of clonal stem cell populations and tumor cells for Hoechst dye uptake.     

High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 μM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.     

A rapid,enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues

A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD⁺)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 ± 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the cuvette concentration range of 0.1 μm to 0.1 mm.