Purification and analysis of a protein cocktail capable of scavenging cell-free hemoglobin, heme, and iron.

BACKGROUND: Hemolysis releases toxic cell-free hemoglobin (Hb), heme, and iron, which overwhelm their natural scavenging mechanisms during acute or chronic hemolytic conditions. This study describes a novel strategy to purify a protein cocktail containing a comprehensive set of scavenger proteins for potential treatment of hemolysis byproducts. STUDY DESIGN AND METHODS: Tangential flow filtration was used to purify a protein cocktail from Human Cohn Fraction IV (FIV). A series of in vitro assays were performed to characterize composition and biocompatibility. The in vivo potential for hemolysis byproduct mitigation was assessed in a hamster exchange transfusion model using mechanically hemolyzed blood plasma mixed with the protein cocktail or a control colloid (dextran 70 kDa). RESULTS: A basis of 500 g of FIV yielded 62 ± 9 g of a protein mixture at 170 g/L, which bound to approximately 0.6 mM Hb, 1.2 mM heme, and 1.2 mM iron. This protein cocktail was shown to be biocompatible in vitro with red blood cells and platelets and exhibits nonlinear concentration dependence with respect to viscosity and colloidal osmotic pressure. In vivo assessment of the protein cocktail demonstrated higher iron transport to the liver and spleen and less to the kidney and heart with significantly reduced renal and cardiac inflammation markers and lower kidney and hepatic damage compared to a control colloid. DISCUSSION: Taken together, this study provides an effective method for large-scale production of a protein cocktail suitable for comprehensive reduction of hemolysis-induced toxicity.

Quantification and Carbon and Nitrogen Isotopic Measurements of Heme B in Environmental Samples.

Heme B is an iron-coordinated tetrapyrrole molecule that acts as a cofactor in hemoproteins. It is expected to be ubiquitous in the environment, as b-type hemoproteins catalyze a variety of essential biochemical reactions. In this study, we developed an analytical method to quantify heme B in biological and environmental samples using high-performance liquid chromatography (HPLC) coupled to a photodiode array detector. The applicability of our method was further extended by the use of liquid chromatography/mass spectrometry (LC/MS; detection limit: ∼1 fmol), which enabled the quantification of a trace amount of dissolved heme B in filtered seawater and sedimentary heme B coexisting with an abundant interfering organic matrix. For compound-specific carbon and nitrogen isotopic measurements, heme B was successfully isolated and purified from biological and environmental samples by a combination of anion-exchange column chromatography, methyl esterification, and dual-step HPLC. While carbon and nitrogen isotopic compositions of heme B in phototrophs were mostly comparable to those of chlorophyll a, heme B in suspended particulate materials in coastal water and an intertidal sediment was 13C-depleted and 15N-enriched relative to chlorophyll a, suggesting that nonphototrophic microorganisms are also a significant source of heme B in natural environments.

Novel manufacturing method for producing apohemoglobin and its biophysical properties.

Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, -80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and ß subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O2 equilibrium curve, O2 dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.

Nutrients Composition in Fit Snacks Made from Ostrich, Beef and Chicken Dried Meat.

The aim of the study was to compare three types of meat snacks made from ostrich, beef, and chicken meat in relation to their nutrients content including fat, fatty acids, heme iron, and peptides, like anserine and carnosine, from which human health may potentially benefit. Dry meat samples were produced, from one type of muscle, obtained from ostrich (m.ambiens), beef (m. semimembranosus), and broiler chicken meat (m. pectoralis major). The composition of dried ostrich, beef, and chicken meat, with and without spices was compared. We show that meat snacks made from ostrich, beef, and chicken meat were characterized by high concentration of nutrients including proteins, minerals (heme iron especially in ostrich, than in beef), biologically active peptides (carnosine-in beef, anserine-in ostrich then in chicken meat). The, beneficial to human health, n-3 fatty acids levels differed significantly between species. Moreover, ostrich jerky contained four times less fat as compared to beef and half of that in chicken. In conclusion we can say that dried ostrich, beef, and chicken meat could be a good source of nutritional components.

Probing Bis-FeIV MauG: Isolation of Highly Reactive Radical Intermediates.

MauG is a diheme enzyme that utilizes two covalently bound c-type heme centers. We report here step-wise oxidations of a synthetic analogue of MauG in which two heme centers are bridged covalently through a flexible linker containing a pyrrole moiety. One- and two-electron oxidations produce monocation radical and dication diradical intermediates, respectively, which, being highly reactive, undergo spontaneous intramolecular rearrangement involving the pyrrole bridge itself to form indolizinium-fused chlorin-porphyrin and spiro-porphyrinato heterodimers. Unlike in MauG, where the two oxidizing equivalents produce the bis-FeIV redox state, the synthetic analogue of the same, however, stabilizes two ferric hemes, each coupled with a porphyrin π-cation radical. The present study highlights the possible role played by the bridge in the electronic communication.

Three phases in pH dependent heme abstraction from myoglobin.

The extent of heme extraction from myoglobin (Mb) by methylethyl ketone is found to be pH dependent and show three distinct phases. Parallel investigations of the protein using resonance Raman (rR) and circular dichroism (CD) across these pH regions indicate that these phases correspond to three different protonation steps in holoMb as the pH of the solution changed. The first transition occurs between pH5-6 and is due to the protonation of one of the heme propionate groups which disrupts its H-bonding with Arg 45 in the loop. The 2nd phase (pH5-4) likely involves the protonation of the 2nd propionate which H-bonds to Ser 92 in the F-helix. The third phase (pH<3.5) involves dissociation of the FeIIHis bond which eventually leads to complete heme dissociation and unfolding.

Mechanism of Human Apohemoglobin Unfolding.

Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α1ß1 interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the ß and γ heme pockets have similar stabilities, whereas the α1γ1 interface is more resistant to dissociation than the α1ß1 interface.

Heme Derived from Corynebacterium glutamicum: A Potential Iron Additive for Swine and an Electron Carrier Additive for Lactic Acid Bacterial Culture.

To investigate the potential applications of bacterial heme, aminolevulinic acid synthase (HemA) was expressed in a Corynebacterium glutamicum HA strain that had been adaptively evolved against oxidative stress. The red pigment from the constructed strain was extracted and it exhibited the typical heme absorbance at 408 nm from the spectrum. To investigate the potential of this strain as an iron additive for swine, a prototype feed additive was manufactured in pilot scale by culturing the strain in a 5 ton fermenter followed by spray-drying the biomass with flour as an excipient (biomass: flour = 1:10 (w/w)). The 10% prototype additive along with regular feed was supplied to a pig, resulting in a 1.1 kg greater increase in weight gain with no diarrhea in 3 weeks as compared with that in a control pig that was fed an additive containing only flour. To verify if C. glutamicum-synthesized heme is a potential electron carrier, lactic acid bacteria were cultured under aerobic conditions with the extracted heme. The biomasses of the aerobically grown Lactococcus lactis, Lactobacillus rhamosus, and Lactobacillus casei were 97%, 15%, and 4% greater, respectively, than those under fermentative growth conditions. As a potential preservative, cultures of the four strains of lactic acid bacteria were stored at 4°C with the extracted heme and living lactic acid bacterial cells were counted. There were more L. lactis and L. plantarum live cells when stored with heme, whereas L. rhamosus and L. casei showed no significant differences in live-cell numbers. The potential uses of the heme from C. glutamicum are further discussed.

The structure, function and properties of sirohaem decarboxylase--an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway.

Some bacteria and archaea synthesize haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side-chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to give didecarboxysirohaem. Further modifications catalysed by two related radical SAM enzymes, AhbC and AhbD, transform didecarboxysirohaem into Fe-coproporphyrin III and haem respectively. The characterization of sirohaem decarboxylase is reported in molecular detail. Recombinant versions of Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Methanosarcina barkeri AhbA/B have been produced and their physical properties compared. The D. vulgaris and M. barkeri enzyme complexes both copurify with haem, whose redox state influences the activity of the latter. The kinetic parameters of the D. desulfuricans enzyme have been determined, the enzyme crystallized and its structure has been elucidated. The topology of the enzyme reveals that it shares a structural similarity to the AsnC/Lrp family of transcription factors. The active site is formed in the cavity between the two subunits and a AhbA/B-product complex with didecarboxysirohaem has been obtained. A mechanism for the decarboxylation of the kinetically stable carboxyl groups is proposed.

Purification, crystallization and phase determination of the DR1998 haem b catalase from Deinococcus radiodurans.

The protective mechanisms of Deinococcus radiodurans against primary reactive oxygen species involve nonenzymatic scavengers and a powerful enzymatic antioxidant system including catalases, peroxidases and superoxide dismutases that prevents oxidative damage. Catalase is an enzyme that is responsible for the conversion of H2O2 to O2 and H2O, protecting the organism from the oxidative effect of H2O2. This study reports the purification and crystallization of the DR1998 catalase from D. radiodurans. The crystals diffracted to 2.6 Å resolution and belonged to space group C2221, with unit-cell parameters a = 97.33, b = 311.88, c = 145.63 Å, suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by molecular replacement and the obtained solution shows the typical catalase quaternary structure. A preliminary model of the protein structure has been built and refinement is currently in progress.

Modulation of the intermolecular interaction of myoglobin by removal of the heme.

Toward understanding intermolecular interactions governing self-association of proteins, the present study investigated a model protein, myoglobin, using a small-angle X-ray scattering technique. It has been known that removal of the heme makes myoglobin aggregation-prone. The interparticle interferences of the holomyoglobin and the apomyoglobin were compared in terms of the structure factor. Analysis of the structure factor using a model potential of Derjaguin-Laudau-Verwey-Overbeek (DLVO) suggests that the intermolecular interaction potential of apomyoglobin is more attractive than that of holomyoglobin at short range from the protein molecule.

Evaluation of unbound free heme in plant cells by differential acetone extraction.

Heme functions not only as a prosthetic group of hemoproteins but also as a regulatory molecule, suggesting the presence of 'free' heme. Classically, total non-covalently bound heme is extracted from plant samples with acidic acetone after removal of pigments with basic and neutral acetone. Earlier work proposed that free heme can be selectively extracted into basic acetone. Using authentic hemoproteins, we confirmed that acidic acetone can quantitatively extract heme, while no heme was extracted into neutral acetone. Meanwhile, a certain amount of heme was extracted into basic acetone from hemoglobin and myoglobin. Moreover, basic acetone extracted loosely bound heme from bovine serum albumin, implying that the nature of hemoproteins largely influences heme extraction into basic acetone. Using a highly sensitive heme assay, we found that basic and neutral acetone can extract low levels of heme from plant samples. In addition, neutral acetone quantitatively extracted free heme when it was externally added to plant homogenates. Furthermore, the level of neutral acetone-extractable heme remained unchanged by precursor (5-aminolevulinic acid) feeding, while increased by norflurazon treatment which abolishes chloroplast biogenesis. However, changes in these heme levels did not correlate to genomes uncoupled phenotypes, suggesting that the level of unbound free heme would not affect retrograde signaling from plastids to the nucleus. The present data demonstrate that the combination of single-step acetone extraction following a sensitive heme assay is the ideal method for determining total and free heme in plants.

Isolation and characterization of free haem from the shell gland of quail and hen.

Free haem was isolated from the shell gland of the quail, Coturnix coturnix japonica, and of the fowl, Galinus domesticus, and characterized by HPLC-ESI-MS/MS. Quantification by HPLC gave values of 1.17-1.40 nmol/mg quail shell gland protein for haem, 1.66-2.17 nmol/mg protein for protoporphyrin and 0.25-0.40 nmol/mg protein for biliverdin. Possible implications of this previously unreported finding are discussed but they are not considered incompatible with the conclusion that all eggshell pigments are endogenously synthesized in the oviduct system.

Methods for analysis of photosynthetic pigments and steady-state levels of intermediates of tetrapyrrole biosynthesis.

Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.

Bioavailability of a heme-iron concentrate product added to chocolate biscuit filling in adolescent girls living in a rural area of Mexico.

A heme-iron concentrate product derived from swine hemoglobin was used to enrich the chocolate-flavored filling of biscuits and the bioavailability of this source of heme-iron was assessed in adolescent girls. The placebo control (PC) group consisted of 35 teenagers with the highest baseline hemoglobin concentrations. The supplemented groups were randomized to receive biscuits fortified with iron sulfate (IS, n = 37) or heme-iron concentrate (HIC, n = 40). Both groups were supplemented with 10.3 mg Fe/d for 7 wk. Blood chemistry and hematology analyses were performed at baseline and at the end of the study. The baseline prevalence of anemia (hemoglobin <12 g/dl) in the entire group was 3.9% and by the end of the study it had fallen to 2.3%. The hemoglobin levels in both supplemented groups increased (P < 0.05) during the study period from 13.6 and 13.5 g/dl for HIC and IS, respectively, at baseline to 14 g/dl at the end of the study. Serum ferritin concentrations decreased by the end of the study in both the PC and IS groups (P < 0.05), but not in the heme group. In conclusion, iron bioavailability from HIC-fortified biscuits was calculated to be 23.7% higher than that observed for IS, as shown by the differences observed in serum ferritin levels during the study. The iron contained in the heme-iron concentrate was well absorbed and tolerated by the adolescents included in the study.

[Glycosaminoglycanes of cartilage tissue of rats after the mechanical trauma: the influence of low-molecular fraction from the cord blood and blood of dairy calves].

Influence of low-molecular fraction (to 5 kDa) from cord blood (FCB) compared with "Actovegin" derived from the blood of veal calves on the content of the matrix polysaccharide components from and proteins of the cartilage connecting tissue after the mechanical trauma was investigated. It is established, that intramuscular administration of FCB essentially stimulates the accumulation of the basic components of the matrix: hexosamine, uronic acid, hyaluronic acid, chondroitinsulfate and heparin, and also the most important components of structural proteins in the cartilage regenerate of the rat knee joint.

A conserved haem redox and trafficking pathway for cofactor attachment.

A pathway for cytochrome c maturation (Ccm) in bacteria, archaea and eukaryotes (mitochondria) requires the genes encoding eight membrane proteins (CcmABCDEFGH). The CcmABCDE proteins are proposed to traffic haem to the cytochrome c synthetase (CcmF/H) for covalent attachment to cytochrome c by unknown mechanisms. For the first time, we purify pathway complexes with trapped haem to elucidate the molecular mechanisms of haem binding, trafficking and redox control. We discovered an early step in trafficking that involves oxidation of haem (to Fe(3+)), yet the final attachment requires reduced haem (Fe(2+)). Surprisingly, CcmF is a cytochrome b with a haem never before realized, and in vitro, CcmF functions as a quinol:haem oxidoreductase. Thus, this ancient pathway has conserved and orchestrated mechanisms for trafficking, storing and reducing haem, which assure its use for cytochrome c synthesis even in limiting haem (iron) environments and reducing haem in oxidizing environments.

Functionally distinct NEAT (NEAr Transporter) domains within the Staphylococcus aureus IsdH/HarA protein extract heme from methemoglobin.

The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. The IsdH protein (also known as HarA) is a receptor for hemoglobin (Hb), haptoglobin (Hp), and the Hb-Hp complex. It contains three NEAT (NEAr Transporter) domains: IsdH(N1), IsdH(N2), and IsdH(N3). Here we show that they have different functions; IsdH(N1) binds Hb and Hp, whereas IsdH(N3) captures heme that is released from Hb. The staphylococcal IsdB protein also functions as an Hb receptor. Primary sequence homology to IsdH indicates that it will also employ functionally distinct NEAT domains to bind heme and Hb. We have used site-directed mutagenesis and surface plasmon resonance methods to localize the Hp and Hb binding surface on IsdH(N1). High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the beta-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.

Heme content of recombinant catalase from Psychrobacter sp. T-3 altered by host Escherichia coli cell growth conditions.

The catalase gene of Psychrobacter sp. T-3 was cloned, and the gene product (PktA) was overexpressed in Escherichia coli. The specific activity of the purified PktA was slightly lower than that of the native purified enzyme obtained from Psychrobacter sp. T-3. Spectrophotometric measurements of the purified enzymes suggested that the recombinant PktA contains a mixture of heme b and d, although the native enzyme contains the sole heme b. An addition of the heme precursor 5-aminolevulinic acid (ALA) to the medium increased the heme b content of the recombinant PktA, and the resulting enzyme showed higher specific activity than the native enzyme. This is the first report that shows the heme content of overproduced catalase altered by the host cell growth conditions.