SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Allhorn, M. Articles by Åkerström, B. Search for Related Content PubMed PubMed Citation Articles by Allhorn, M. Articles by Åkerström, B. Related Collections Red Cells Plenary Papers Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 March 2002, Vol. 99, No. 6, pp. 1894-1901 PLENARY PAPER Processing of the lipocalin 1-microglobulin by hemoglobin induces heme-binding and heme-degradation properties Maria Allhorn, Tord Berggård, Jonas Nordberg, Martin L. Olsson, and Bo Åkerström From the Department of Cell and Molecular Biology, Lund University, and Department of Transfusion Medicine, Blood Center, University Hospital, Lund, Sweden.     Abstract Top Abstract Introduction Materials and methods Results Discussion References 1-Microglobulin is a 26-kd protein, widespread in plasma and tissues and well-conserved among vertebrates. 1-Microglobulin belongs to the lipocalins, a protein superfamily with highly conserved 3-dimensional structures, forming an internal ligand binding pocket. The protein, isolated from urine, has a heterogeneous yellow-brown chromophore bound covalently to amino acid side groups around the entrance of the lipocalin pocket. 1-Microglobulin is found in blood both in free form and complex-bound to immunoglobulin A (IgA) via a half-cystine residue at position 34. It is shown here that an 1-microglobulin species, which we name t-1-microglobulin (t = truncated), with a free Cys34 thiol group, lacking its C-terminal tetrapeptide, LIPR, and with a more polar environment around the entrance of the lipocalin pocket, is released from IgA-1-microglobulin as well as from free 1-microglobulin when exposed to the cytosolic side of erythrocyte membranes or to purified oxyhemoglobin. The processed t-1-microglobulin binds heme and the 1-microglobulin-heme complex shows a time-dependent spectral rearrangement, suggestive of degradation of heme concomitantly with formation of a heterogeneous chromophore associated with the protein. The processed t-1-microglobulin is found in normal and pathologic human urine, indicating that the cleavage process occurs in vivo. The results suggest that 1-microglobulin is involved in extracellular heme catabolism. (Blood. 2002;99:1894-1901) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Hemoglobin, the major oxygen carrier system in the blood, has a number of toxic, potentially dangerous side effects.1,2 Most of these have their origin in the auto-oxidation of oxyhemoglobin. Hemoglobin is a tetramer consisting of 4 globin subunits (22), each carrying a heme group in its active center.3 Heme consists of protoporphyrin IX and a ferrous (Fe2+) iron atom which has high affinity for free oxygen (O2). Ferrous hemoglobin binding to O2 is called oxyhemoglobin. Auto-oxidation of oxyhemoglobin is a spontaneous intramolecular oxidation-reduction reaction eventually leading to production of ferric (Fe3+) hemoglobin (methemoglobin), ferryl (Fe4+) hemoglobin, free heme ("heme" and "hemin" are sometimes used to designate free protoporphyrin IX with a bound Fe2+ or Fe3+ atom, respectively; in this article, "heme" is used regardless of the iron oxidation state), and various reactive oxygen species, including free radicals.4 These compounds present considerable oxidative stress, leading to tissue damage and cell destruction. The overwhelming part of hemoglobin is found strictly compartmentalized within erythrocytes. The auto-oxidation of oxyhemoglobin and downstream free-radical formation is largely prevented by the intracellular inhibitors superoxide dismutase, catalase, and glutathione peroxidase.5,6 In spite of this, slow auto-oxidation occurs intracellularly. Oxidized hemoglobin forms which are unstable and easily denatured are found deposited together with free heme and iron on the cytosolic face of the erythrocyte membrane.7 Hemoglobin is also found extracellularly in plasma at normal concentrations up to around 5 mg/L, mainly as a result of hemolysis.8 Plasma contains haptoglobin,9 a high-affinity binder of oxyhemoglobin and inhibitor of auto-oxidation; iron- and heme-binding proteins such as transferrin, albumin, and hemopexin; and antioxidants such as vitamin E and ascorbic acid. However, it is generally agreed that these systems are not sufficient to protect against hemoglobin-mediated oxidative cell and tissue damage during increased extravascular hemolysis and general hemolytic pathologic disorders. 1-Microglobulin (1m) is a plasma and tissue protein with unknown biologic functions. It is evolutionarily well-conserved and has so far been found in mammals, birds, fish, and amphibians. 1m is a glycoprotein with a relatively low molecular mass, 26 kd.10 Hence, it is filtered through the glomerular membranes of the kidney and was originally isolated from urine.11 1m, also called protein HC,12 has many intriguing properties.13,14 It belongs to the lipocalin superfamily, a group of proteins from animals, plants, and bacteria with a well-conserved 3-dimensional structure. The lipocalins consist of a 160 to 190 amino-acid polypeptide folded into an 8-stranded -barrel surrounding a pocket that can bind hydrophobic ligands.15 The ligand of 1m is unidentified. 1m carries an extremely heterogeneous yellow-brown chromophore, covalently bound to lysyl residues (Lys 92, 118, and 130) surrounding the entrance of the lipocalin pocket.16 1m has one unpaired cysteine residue (Cys 34) which can interact with a second free Cys exposed at the surface of other proteins. Thus, a circulating covalent complex between 1m and immunoglobulin A (IgA), involving about 50% of plasma-1m, was shown to be mediated by Cys 34 of 1m and the penultimate Cys residue of the IgA -chain.17 The Cys-Cys interchain cross-link of IgA-1m has so far been impossible to reduce in vitro. Therefore, the original observation of this work, the cleavage of IgA-1m into free 1m and IgA by ruptured erythrocytes, was surprising. It prompted an investigation of the role of 1m in hemolysis. The results indicate that an activated form of 1m, which participates in degradation of heme, is released from free 1m and its IgA complex by exposed erythrocyte membranes and hemoglobin. The results suggest a possible biologic function of 1m in protection against unsequestered heme/hemoglobin that could explain its widespread distribution in tissues and species as well as some of its enigmatic biochemical properties.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Proteins and reagents Urine and blood from apparently healthy donors and urine from patients were collected. Information about the medical condition and serologic data for the patient and control groups was obtained from the Hematology Department and Blood Center at the University Hospital in Lund, Sweden. Approval was obtained from the institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki. Blood was drawn with addition of heparin anticoagulant. The urine and plasma, prepared from the blood, were frozen at 30°C within 1 hour. Oxyhemoglobin was prepared by diethylaminoethyl (DEAE)-Sephadex ion-exchange chromatography of erythrocyte hemolysates according to Winterbourn.4 IgA-1m, plasma-1m, urine 1m, and pepsin-1m were purified in our laboratory as described earlier.16,18,19 Rabbit anti-1m was prepared by immunization with urine 1m as described.20 Rabbit anti-LIPR was prepared by AgriSera AB (Vännäs, Sweden) by immunization with the synthetic peptide CKKLIPR conjugated to keyhole limpet hemocyanin (KLH). Mouse monoclonal anti-1m, BN11.10, was prepared and purified as described21 and immobilized to Affigel Hz (Bio-Rad Labs, Hercules, CA) at 20 mg/mL, following instructions from the manufacturer. Goat antihuman heme oxygenase I (cat. no. sc-1796) was from Santa Cruz Biotechnology (Santa Cruz, CA). Orosomucoid, ovalbumin, and human serum albumin were purchased from Sigma Chemicals (St Louis, MO). Erythrocytes and membranes Blood was centrifuged at 1200g for 10 minutes, plasma- and buffy coat-aspirated, and the red blood cells washed 4 times with 10 volumes of phosphate buffered saline (PBS): 10 mM phosphate buffer, pH 7.4, 120 mM NaCl, 3 mM KCl. The packed erythrocytes were lysed by resuspension in 1 volume of cold, hypotonic buffer (H2O:PBS, 20:1) on ice. Membrane pellets were separated from cytosol by centrifuging the lysed suspension at 14 000g for 20 minutes with subsequent washing. The pellets were then resuspended in PBS to one-tenth of the original blood volume. Hemoglobin-free membranes were prepared by repeated washing as described by Dodge et al.22 Membrane proteins were quantitated by the BCA Protein Assay Kit (Pierce, Rockford, IL). For further fractionation of membrane constituents, the lysed erythrocytes were centrifuged at 32 000g for 30 minutes, the membrane pellet solubilized in 50 mM Tris-HCl, pH 8.0 + 1% Nonidet P-40, and centrifuged at 8000g for 10 minutes. The supernatant was then applied to Sephacryl S-300 gel chromatography, eluting with 20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, and 0.02% NaN3, and analyzing the eluted fractions for ultraviolet (UV) absorbance and 1m cleavage activity as described below. Cleavage of 1-microglobulin Freeze-dried 1m or IgA-1m was incubated for indicated time periods at 37°C with the lysed, nonfractionated erythrocytes, cytosol, or membranes at a final 1m concentration of 40 µM. For cleavage with purified oxyhemoglobin, 0.04 µmol freeze-dried plasma-1m was incubated for 3 hours at 37°C with 0.2 µmol oxyhemoglobin tetramer in 0.2 mL PBS. After incubation, 1m was purified from the incubation mixtures by affinity chromatography on monoclonal anti-1m Affigel columns. After washing the columns with PBS, adsorbed 1m was eluted by the addition of 0.1 M glycine-HCl, pH 2.3. Eluted fractions were immediately neutralized with one-tenth volume of 1 M Tris-HCl, pH 8.5, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions. The 1m proteins were sometimes alkylated with iodoacetamide (IAA) before the cleavage, incubating 1 mg/mL protein and 0.17 M IAA in 20 mM Tris-HCl, pH 8.5, for 30 minutes at room temperature and dialysing against PBS. For cleavage of 125I-1m or IgA-1m, 10 µL plasma, lysed erythrocytes, membranes, cytosol, or gel chromatography fractions were incubated with .5 µCi (20 kBq) 125I-proteins 14 µCi (.5 MBq)/µg in PBS. The reaction proceeded for 3 hours at 37°C. One microliter of the incubations was diluted 10 times with PBS and applied to SDS-PAGE. Time-dependence of 1m cleavage A quantity of 0.5 µg of plasma-1m was added to 4 µL lysed erythrocytes or 15 µL erythrocyte membranes. In both cases the total sample volume was adjusted to 20 µL with PBS. The samples were incubated at 37°C for 1, 5, 30, 60, and 240 minutes. Cleavage was determined by Western blotting using antibodies against 1m and LIPR. Heme binding experiments The binding of heme by 1m was studied by incubation with (1) methemoglobin-Sepharose, (2) purified oxyhemoglobin, and (3) [14C]heme. Oxyhemoglobin was immobilized to CNBr-activated Sepharose 4B at 10 mg/mL (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the description from the manufacturer and with modifications made by Gattoni et al.23 The immobilized oxyhemoglobin was oxidized to the ferric form (methemoglobin) by incubating with 1.2 equivalents of potassium ferricyanide (K3Fe[CN]6) at room temperature for 30 minutes and then washed extensively with 0.1 M phosphate buffer, pH 7.4. Methemoglobin-Sepharose was incubated with 1m or control proteins in PBS. The reaction proceeded for 60 minutes at room temperature with careful agitation and was terminated by pelleting the hemoglobin-Sepharose and removing the supernatants for absorbance spectrum analysis. Oxyhemoglobin (7 µM) and 1m or control proteins (5 µM) were incubated in PBS at 37°C for 1 hour. 1m was purified by affinity chromatography on a column of monoclonal anti-1m Affigel, concentrated and analyzed by spectrophotometry. 1m or control proteins (0.1 nmol-0.2 nmol) in 10 µL PBS were incubated with 25 pmol [14C]heme diluted in PBS plus 1% Tween-20. The incubation was performed at room temperature in dim light. The reaction was stopped at different time intervals by adding SDS-PAGE sample buffer, separating by SDS-PAGE, staining, and phosphoimaging analysis (see below). Heme degradation Oxyhemoglobin (3 µM) was incubated alone or with t-1m or control proteins (24 µM) in 20 mM phosphate buffer, pH 7.4, at 37°C. The absorbance at 410 nm was read at 5-minute intervals for up to 24 hours, blanking with the reaction solutions themselves at time zero. Preparation of [14C]heme Radiolabeled, 14C-substituted heme was produced using the E coli strain AN344 containing the plasmid pTYR13. This strain/plasmid combination (kindly donated by Dr Lars Hederstedt, Department of Microbiology, Lund University) requires the heme-precursor -aminolevulinic acid (ALA) for growth and was cultured with LB-medium (10 g/L Tryptone-Peptone, Difco, Becton Dickinson, Sparks, MD, 5 g/L yeast extract, Merck, Darmstadt, Germany, 5 g/L sodium chloride) containing [4-14C]ALA, 50 Ci/mol (New England Nuclear, Boston, MA) as described by Schiött et al.24 The yield of [14C]heme was about 20 nmol per 60-mL culture with a specific activity of 2.5 Ci/mmol (90GBq/mmol). Gel electrophoresis and immunoblotting SDS-PAGE was performed using either 10% or 12% slab gels in the buffer system described by Laemmli,25 sometimes including 2% vol/vol -mercaptoethanol in the sample buffers. High-molecular-mass standards (rainbow markers; Amersham Pharmacia Biotech, Uppsala, Sweden) were used. The polyacrylamide gels were stained with Coomassie Brilliant Blue R-250 and in some cases dried. For immunoblotting, the gels were transferred to polyvinylidenefluoride (PVDF) membranes (Immobilon, Millipore, Bedford, MA) as described.26 The membranes were then incubated with antiserum as previously described.27 Radioactive samples (14C or 125I) in dried gels and membranes were analyzed by phosphoimaging in a Fujix BAS 2000 Bioimaging analyzer (Fujifilm Sverige AB, Stockholm, Sweden). Protein radiolabeling Proteins were labeled with 125I (Svensk Radiofarmaka AB, Stockholm, Sweden) using the chloramine-T method.28 The labeled proteins were separated from free iodide by desalting the reaction mixture on prepacked 9-mL Sephadex G-25 columns (PD10, Amersham Pharmacia Biotech). The specific activity of the labeled proteins was approximately 14 µCi(0.5 MBq)/µg protein. Spectrophotometric methods Absorbance spectra were measured on a Beckman (Beckman Instruments, Fullerton, CA) DU 640i spectrophotometer using a scan rate of 240 nm/minute and protein concentrations between 1 µM and 50 µM. Fluorescence spectra were recorded with a Perkin Elmer (Sollentuna, Sweden) LB50 fluorescence spectrometer. The excitation was made at 280 nm with an excitation slit-width of 5 nm and an emission slit-width of 3 nm. The scan speed was 150 nm/minute. The proteins used in this experiment were diluted with PBS to a concentration of 4 µM. Carbohydrate analysis of 1-microglobulin Detection of O-linked and N-linked oligosaccharides was done by glycosidase cleavage and lectin blotting as described.18 Amino acid sequence analysis Amino acid sequencing was done by Dr Bo Ek at Statens Lantbruksuniversitet, Uppsala, Sweden. Briefly, proteins were separated by SDS-PAGE, specific bands cut from the gel and digested by trypsin incubation. Trypsin digests were characterized by electrospray tandem mass spectrometry. This made it possible to make identifications based both on peptide mass data as well as on sequence information (peptide fragmentation data). All measurements were made on a Q-tof instrument (Micromass, Manchester, United Kingdom) essentially according to the manufacturer's instructions. Alkylation with iodo[14C] acetamide Proteins were incubated with iodo[14C] acetamide ([14C]IAA) (Amersham Life Science, specific activity 59.0 mCi(2.2 GBq)/mmol). The reaction mixtures contained 4 µM protein in 0.2 M Tris-HCl, pH 8.5, and 1 mM [14C]IAA. The reaction proceeded for 75 minutes at 25°C in the dark. To determine the amounts of bound [14C]IAA, the alkylated proteins were subjected to SDS-PAGE and phosphoimaging. Gel chromatography Proteins were separated by gel chromatography on a 50-mL Sephacryl S-300 column (Pharmacia, Uppsala, Sweden), equilibrated with 20 mM Tris-HCl, 150 mM NaCl, 0.02% NaN3, pH 8.0, at 4°C. The column was eluted at a flow rate 12 mL/hour and the eluted fractions were analyzed by absorbance at 280 nm and 410 nm.     Results Top Abstract Introduction Materials and methods Results Discussion References Cleavage of 1-microglobulin by erythrocyte membranes and hemoglobin Incubation of free plasma-1m with lysed erythrocytes, or the membrane or cytosolic fractions separately, resulted in a reduction in size of the protein when analyzed by SDS-PAGE. The truncated form of 1m, which we henceforth call t-1m, had an apparent molecular mass of 30 kd, approximately 3 kd less than uncleaved 1m (Figure 1). The IgA-1m complex was also cleaved by the erythrocyte fractions. In this complex 1m is covalently cross-linked by a nonreducible disulfide bond to one of the heavy ()-chains of IgA. The IgA-1m complex appears as 3 major bands on SDS-PAGE: the 90-kd 1m--chain, the 60-kd -chain, and the 25-kd light chain. In addition, various less-abundant high-molecular-weight bands can be seen, representing polymeric forms of IgA-1m containing the 90-kd IgA-1m--chain component. After incubation with the erythrocyte fractions, the 90-kd IgA--chain band disappeared, and was replaced by free 1m with a molecular mass of 30 kd, that is, t-1m (Figure 1). The same incubation conditions did not affect the migration of control proteins orosomucoid (Figure 1), human serum albumin, IgG, IgA, and ovalbumin (not shown) upon SDS-PAGE. Cleavage of 1m and IgA-1m was achieved using up to 80 nmol protein/mL erythrocytes. The cleavage could be detected after purification by anti-1m affinity chromatography followed by SDS-PAGE (see below), by Western blotting of the erythrocyte/1m mixture, or by using 125I-labeled protein (see "Materials and methods"). View larger version (78K): [in this window] [in a new window]   Figure 1. Cleavage of 1m and control proteins by erythrocyte membranes. Plasma-1m, IgA-1m, and orosomucoid were incubated with purified erythrocyte membranes for 2 hours at 37°C. A quantity of 1 µg to 3 µg of each protein was separated by SDS-PAGE (T = 12%, C = 3.3%) without prior affinity chromatography purification of the 1m-components and stained with Coomassie Brilliant Blue. Lane 1 shows the proteins alone and lane 2 shows the proteins with added erythrocyte membranes. The electrophoresis was performed in the presence of mercaptoethanol. Molecular masses of standards are given in kilodaltons. Erythrocytes were lysed and separated by centrifugation into membrane and cytosolic fractions that were suspended in the original blood cell volume. As stated above, both fractions cleaved plasma-1m into t-1m. Whole, nonruptured erythrocytes had no cleaving activity at all. Gel filtration on Sephacryl S-300 of solubilized membranes showed that the cleaving activity was associated with medium-sized molecules (not shown). Hemoglobin is a major macromolecular constituent of both the cytosolic and membrane portions of the lysed erythrocytes. Therefore, we also tested cleaving of 1m by purified hemoglobin. Mixing oxyhemoglobin with plasma-1m resulted in the same decrease in size as the erythrocyte fractions (Figure 2A). The specific cleaving activity was estimated by dilution of the membrane and cytosolic fractions and purified hemoglobin (hemoglobin tetramer concentration 0.02 mM, 4 mM, and 1 mM, respectively). The membrane fraction had much higher specific cleaving activity than the other 2; that is, t-1m was formed from plasma-1m by the membranes at more than 10-fold higher dilution than the cytosol or purified hemoglobin. Hemoglobin-free membranes had no cleaving activity (not shown). These results suggest that hemoglobin in synergy with unknown factors in erythrocyte membranes, alternatively a minor form of hemoglobin found enriched in erythrocyte membranes, mediate the cleavage of 1m. View larger version (87K): [in this window] [in a new window]   Figure 2. Release of the C-terminal tetrapeptide from 1m after cleavage with erythrocyte membranes and oxyhemoglobin. Plasma-1m (lanes 1 and 3) was incubated with erythrocyte membranes (lane 2) or oxyhemoglobin (lane 4) as described in "Materials and methods." After incubation for 3 hours at 37°C, 1m was purified from the reaction mixtures by affinity chromatography and gel chromatography on Sephacryl S-300. Purified 1m, 1 µg to 3 µg, was separated by SDS-PAGE in the presence of mercaptoethanol, staining, and Western blotting with antibodies against 1m or LIPR. C-terminal proteolysis of 1-microglobulin The t-1m formed by cleavage of plasma, recombinant, or IgA-1m was purified by applying the mixture of 1m and lysed erythrocytes or hemoglobin to a column of immobilized monoclonal antibodies against 1m followed by gel chromatography on Sephacryl S-300 (Figure 2, lanes 2 and 4, respectively). Hemoglobin was coeluted with 1m from the anti-1m column but was removed by the gel chromatography step. The cleavage of IgA-1m yielded an intact IgA molecule, besides t-1m, as judged by SDS-PAGE (eg, Figure 1) or Sephacryl S-300, underscoring the specificity of the cleavage mechanism. The amino acid sequence of purified t-1m was analyzed by trypsin cleavage followed by mass spectrometry, and it was shown that 1m was cleaved C-terminally; that is, the C-terminal tetrapeptide LIPR (amino acid pos. 180-183) was missing in t-1m whether it was produced from free plasma-1m or IgA-1m, whereas the N-terminus was intact (Table 1). Polyclonal rabbit antibodies were raised against the synthetic peptide LIPR and used to confirm the absence of the tetrapeptide in t-1m. Thus, after SDS-PAGE and blotting, anti-LIPR recognized intact full-length 1m but not t-1m (Figure 2C), whereas a conventional anti-1m antiserum recognized both intact, full-length 1m and t-1m (Figure 2B).                                View this table: [in this window] [in a new window]   Table 1. Molecular composition of 1m before and after cleavage by erythrocyte membranes and purified oxyhemoglobin The time course of the cleavage of plasma-1m by lysed erythrocytes and erythrocyte membranes was studied by SDS-PAGE followed by blotting with anti-1m and anti-LIPR (Figure 3). The results shows that the cleavage by erythrocyte membranes was completed after 5 minutes. View larger version (69K): [in this window] [in a new window]   Figure 3. Time studies on the cleavage of 1m. A quantity of 0.5 µg plasma-1m was incubated with lysed erythrocytes, erythrocyte membranes, or PBS (control) at 37°C for indicated time points: 1, 5, 30, 60, and 240 minutes. The cleavage reaction was stopped by adding SDS-PAGE sample buffer and was analyzed by SDS-PAGE in the presence of mercaptoethanol and Western blotting with anti-1m and anti-LIPR. The results thus suggest that the C-terminal tetrapeptide of 1m is released from plasma-1m and the IgA-1m complex when exposed to hemoglobin and other unknown factors of the cytosol and cytosolic surface of erythrocyte membranes. Biochemical properties of t-1-microglobulin Characterization of purified t-1m from plasma-1m and IgA-1m showed that the cleavage involved the C-terminus (see above), the unpaired Cys34 residue (in the case of IgA-1m), and the chromophores of 1m, whereas the N-terminus as well as the carbohydrate moieties were intact (Table 1). The reactivity of the unpaired sulfhydryl group (-SH) of Cys34 was investigated by alkylation with iodo[14C]acetamide (Figure 4; Table 1). Plasma-1m could be alkylated with iodo[14C]acetamide (Figure 4A, lane 1), indicating that the sulfhydryl group of Cys34 is free. The protein was still alkylable after cleavage (Figure 4A, lane 2). IgA-1m was first alkylated with an excess of nonlabeled iodoacetamide to block all remaining free sulfhydryl groups on the IgA part of the molecule (Figure 4B, lane 2) and then subjected to cleavage by erythrocyte membranes. Free sulfhydryl groups then appeared on both the 1m- and -chain parts (Figure 4B, lane 3). The 1m part of IgA-1m, called pepsin-1m, can be prepared by pepsin cleavage.17 It is composed of the C-terminal nonapeptide of the IgA--chain linked to the intact 1m polypeptide by an unusual, nonreducable bond involving Cys34 of 1m and the penultimate Cys of the -chain.16,17 As expected, pepsin-1m could not be alkylated with iodo[14C]acetamide (Figure 4C, lane 1). However, incubation of pepsin-1m with erythrocyte membranes yielded a free, alkylable sulfhydryl group (Figure 4C, lane 2). The results thus suggest that the cleavage of IgA-1m involves both a reduction of the bond between IgA and 1m and a C-terminal truncation of the 1m part. View larger version (71K): [in this window] [in a new window]   Figure 4. Appearance of free thiol groups on 1m after cleavage with erythrocyte membranes. (A) A quantity of 3 µg free plasma-1m (lane 1) was incubated with erythrocyte membranes and purified by affinity chromatography on a column with anti-1m (lane 2). The samples were treated with [14C]IAA, separated by SDS-PAGE, stained, and analyzed by phosphoimaging. (B) A quantity of 10 µg IgA-1m was either left untreated (lane 1), alkylated with cold IAA (lane 2), or alkylated with cold IAA and incubated with lysed erythrocytes (lane 3). The proteins were then incubated with [14C]IAA, separated by SDS-PAGE and stained, or analyzed by phosphoimaging. (C) The 1m-fragment of IgA-1m was prepared by pepsin digestion. A quantity of 3 µg of the 1m-fragment was either left untreated (lane 1) or incubated with lysed erythrocytes (lane 2). The proteins were incubated with [14C]IAA, separated by SDS-PAGE and stained, or analyzed by autoradiography. All electrophoreses were performed in the presence of mercaptoethanol. The extremely heterogeneous chromophore of 1m gives the protein a characteristic absorbance spectrum with a slowly declining absorbance throughout the upper UV and visible regions. It has been shown previously that urinary 1m carries more chromophoric material than plasma-1m, whereas IgA-1m has almost no chromophore.16,27 Figure 5 shows that t-1m from both plasma- and IgA-1m had a higher absorbance in the upper UV and visible regions as compared with the uncleaved proteins. A shoulder in the absorbance was sometimes seen around 410 nm. t-1m was also brown-colored in the test tube, whereas plasma-1m is light yellow and IgA-1m and pepsin-1m are uncolored at the same concentrations. View larger version (19K): [in this window] [in a new window]   Figure 5. Absorbance spectrum of IgA-1m and free plasma-1m before and after cleavage with lysed erythrocytes. Uncleaved IgA-1m is represented by pepsin-1m (eg, the 1m part of IgA-1m). Approximately 20 µM solutions in PBS were used. 1m contains 4 tryptophan residues, 3 of which are located near the entrance of the lipocalin pocket.29 The tryptophan fluorescence spectra of free plasma-1m, pepsin-1m, and t-1m displayed emission maxima at 337, 338, and 345 nm, respectively (not shown). This shows that the local environment around the 4 tryptophan residues is more polar in t-1m as compared with free plasma-1m and pepsin-1m (ie, the 1m part of IgA-1m). The carbohydrate portion of 1m, 2 N-linked and one O-linked oligosaccharide,30 was intact after the cleavage as determined by investigation with specific endoglycosidases and lectins (Table 1). Cleaved 1-microglobulin in urine Due to its small size, 1m is passed from the blood to the primary urine via glomerular filtration. A small part escapes tubular reabsorption and is excreted in the urine. It was shown that normal urine contains t-1m, lacking C-terminal LIPR, and full-length 1m (Figure 6, lanes 4-6). Furthermore, an increased t-1m fraction was seen in urine from patients with hemolytic disorders (Figure 6, lanes 1-3), suggesting a relationship to pathologic erythrocyte destruction. This indicates that the cleavage of 1m is a normally occurring biologic process and that t-1m may be produced during hemolysis in vivo. View larger version (40K): [in this window] [in a new window]   Figure 6. SDS-PAGE of urinary 1m. Urinary 1m was purified by affinity chromatography on a column with monoclonal antibodies against 1m. The eluate was analyzed by SDS-PAGE in the presence of mercaptoethanol (T = 12%, C = 3,3), staining, and Western blotting with anti-1m and anti LIPR. Urine from apparently healthy donors (lanes 4-6) as well as patients having different disorders associated with the following changes in erythrocyte formation and destruction were used: lane 1, suspected mechanical hemolytic anemia associated with mitral valve reconstruction following endocarditis; lane 2, autoimmune hemolytic anemia (warm type, IgG-mediated); lane 3, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome. Binding and degradation of heme The shoulder in the absorbance spectrum of freshly prepared t-1m (Figure 5) even after gel chromatography suggests the presence of heme, which has a characteristic absorbance peak at 405 nm to 415 nm called the Soret band. This prompted an investigation of a possible interaction among 1m, t-1m, and heme. Equimolar amounts of plasma-1m, t-1m, human serum albumin, and orosomucoid were incubated with [14C]heme for various time periods, separated by SDS-PAGE, and analyzed by autoradiography (Figure 7). Both plasma-1m and t-1m displayed a binding of radiolabeled heme. Interestingly, more heme was bound to 1m, especially to t-1m, than to human serum albumin, a physiologic heme-binding protein.31 Orosomucoid, a negative control from the Lipocalin superfamily, did not show any binding. View larger version (87K): [in this window] [in a new window]   Figure 7. Time-dependence of [14C]heme-binding to 1m. Approximately 0.2 nmol plasma-1m, t-1m, human serum albumin (HSA), and orosomucoid was incubated with 25 pmol [14C]heme for 1, 3, 15, 60, and 180 minutes. The binding of [14C]heme to the proteins was then determined by SDS-PAGE, staining (A), and phosphoimaging (B). The heme binding was also investigated using hemoglobin insolubilized to Sepharose and oxidized to methemoglobin. Methemoglobin-Sepharose has been shown to bind heme less tightly than oxyhemoglobin and the metheme-group is transferred to albumin and other heme-binding proteins.23 Plasma-1m and t-1m were incubated with methemoglobin-Sepharose and the absorbance spectrum of the supernatants showed a binding of metheme to both proteins (Figure 8A,D). The binding of heme was seen as a pronounced Soret band for both plasma-1m and t-1m. However, the time-dependence of the binding was radically different between the 2 1m-forms using this method. The proteins were incubated with methemoglobin-Sepharose for one hour, removed by centrifugation and their absorbance spectra then analyzed at different times. After 40 hours the Soret band of heme had disappeared almost completely from t-1m (Figure 8A). Instead, the absorbance at lower wavelengths increased (300 nm-400 nm), yielding a heterogeneous spectrum that resembles the highly brown-colored urinary 1m and recombinant 1m from baculovirus-infected insect cells.27 On the other hand, the absorbance spectrum of free plasma-1m was stable during this time period (Figure 8D). The rearrangement of the spectrum was also observed when t-1m was incubated with soluble oxyhemoglobin (Figure 8B). Spectral analysis of t-1m freshly isolated after incubation of plasma-1m with lysed erythrocytes also revealed a decrease of the Soret band concomitant with an increase at 300 nm to 400 nm (Figure 8C). These results suggest a simultaneous heme degradation and chromophore formation in t-1m. View larger version (21K): [in this window] [in a new window]   Figure 8. Spectral analysis of t-1m after incubation with hemoglobin. (A) Methemoglobin-Sepharose (75 µM) and t-1m (3 µM) were incubated in PBS at 37°C for 1 hour. The sample was centrifuged and the absorbance spectrum of the supernatant measured at the indicated time intervals. The spectrum of a control sample (methemoglobin-Sepharose only) was subtracted. (B) Oxyhemoglobin (7 µM) and t-1m (5 µM) were incubated in PBS at 37°C for 1 hour. The t-1m was purified by affinity chromatography on a column of monoclonal anti-1m Affigel, concentrated, and the absorbance spectrum read after 0 and 5 hours. (C) Lysed erythrocytes (original cell volume diluted 1:1) and plasma-1m (40 µM) were mixed and incubated at 37°C for 3 hours. The t-1m formed was purified by affinity chromatography on a column of monoclonal anti-1m Affigel and gel chromatography on Sephacryl S-300, concentrated, and the absorbance spectrum read after the indicated time intervals. (D) Methemoglobin-Sepharose (75 µM) and plasma-1m (10 µM) were incubated for 1 hour at 37°C. The sample was centrifuged and the absorbance spectrum of the supernatant measured at the indicated time intervals. Figure 8B shows that heme is transferred from oxyhemoglobin to t-1m and degraded. The time-dependence of the degradation was investigated more closely by continuously reading the absorbance at 410 nm in a t-1m and oxyhemoglobin mixture using an excess of 1m (molar ratio 8:1). Figure 9 shows that most of the absorbance at 410 nm disappeared within one hour, suggesting that the degradation is a rapid process. View larger version (15K): [in this window] [in a new window]   Figure 9. Rate of heme-degradation in a t-1m and oxyhemoglobin mixture. Oxyhemoglobin (3 µM) was incubated alone or mixed with t-1m (24 µM) at 37°C in 0.5 mL 20 mM phosphate buffer, pH 7.4. The absorbance at 410 nm was read at 5-minute intervals for 10 hours, blanking with the incubation solution itself at time zero.     Discussion Top Abstract Introduction Materials and methods Results Discussion References The results in this work show that plasma-1m is proteolytically processed by ruptured erythrocytes and the C-terminal tetrapeptide LIPR is released. The cleavage is induced by hemoglobin and factors in erythrocyte membranes. The released, processed 1m (t-1m) binds and degrades heme with a concomitant formation of a yellow-brown chromophore strongly linked to the protein. The results thus suggest that 1m has a role in heme catabolism after exposure of hemoglobin and erythrocyte membranes. The processed t-1m was found in urine together with the unprocessed form. In blood, 1m is rapidly lost from the circulation by glomerular filtration.32 Therefore, the t-1m found in urine most likely has been filtered from plasma. This suggests that the cleavage described in this paper actually occurs in vivo. The finding is supported by a previous report that urinary 1m is a mixture of full-length 1m with 183 amino acids and a C-terminally truncated 179-amino acid form.33 The proportion of t-1m in urine should reflect to what extent 1m is cleaved C-terminally in blood and/or tissues. It is therefore of interest to measure the ratio of full-length 1m/t-1m in urine of patients with for example hemolytic disorders as compared with healthy individuals. An initial approach to such an investigation is shown in Figure 6. As expected, the ratio varies individually but a relatively higher amount of t-1m is indicated in 2 of the 3 patients. Indeed, 1m from the patient with mainly extravascular hemolysis is almost completely cleaved. Although it must be emphasized that this is not a clinical study, the preliminary findings are encouraging. Larger patient groups as well as a methodological fine-tuning are needed to better evaluate the potential clinical applications of the t-1m/1m ratio. Interestingly, the chromophore of urinary 1m has a much more pronounced yellow-brown color and absorbance spectrum than plasma-1m. The explanation for this could be that urinary 1m partly consists of t-1m, since it was shown here that the absorbance spectrum and color of the latter is similar to urinary 1m. The C-terminal processing of 1m is apparently performed by factors found inside the erythrocytes. No sign of transport of 1m across the erythrocyte membranes has been detected (not shown). It must therefore be concluded that in vivo processing only takes place after rupture of the red blood cells and exposure of the interior of the cell to 1m. Purified hemoglobin displayed a processing activity at high concentrations and may be responsible for the cleavage activity found in the cytosol, where it is found in very high amounts. The membrane fraction showed a stronger specific processing activity than the cytosolic fraction. Hemoglobin is found deposited in various aggregated and oxidized forms on the cytosolic face of the erythrocyte membranes.7 Therefore, it is possible that a variant of hemoglobin and other unknown membrane factors cause the C-terminal processing of 1m. The results suggest that the processed t-1m induces a degradation of heme which is accompanied by a chromophore formation in the protein. It has been shown that the chromophore consists of covalent modifications of Lys 92, 118, and 130. According to molecular modeling of 1m these residues are located semiburied at the entrance of the lipocalin pocket.16,29 It is possible that heme binds to the lipocalin pocket and induces the chromophore formation either as a direct precursor or by a reaction mechanism in which the chromophore is a by-product. Fluorescence analysis suggests that the tryptophan residues are more exposed to the hydrophilic environment in t-1m. Interestingly, 3 of the 4 tryptophan residues are located around the entrance of the lipocalin pocket. It may be speculated that the C-terminal tetrapeptide is located close to the entrance of the pocket and that its proteolytic removal initiates heme degradation by exposing reactive side groups. IgA-1m may be regarded as a depot of 1m from which 1m is released and processed locally as soon as the erythrocyte ruptures. The size of IgA-1m prevents the molecule from glomerular filtration and loss from the circulation. The release of 1m from IgA and the C-terminal processing are probably 2 separate reactions requiring separate cofactors. The former reaction involves a reduction of the disulfide bond between Cys34 on 1m and the Cys residue on the -chain. The bond is unusually reduction-resistant and it has previously not been possible to break the bind in vitro. It was shown here that Cys34 on t-1m is reduced and presents a free thiol group. According to the structural 1m-model this residue is exposed and located on a flexible omega-loop near the entrance to the pocket. Cys34 has previously been shown to be attached to chromophore substances,34 and it is possible that it is involved in the heme-binding and chromophore formation. Other heme-binding proteins have been shown to bind the heme group via an unpaired Cys residue; for instance, proteins carrying a so-called heme regulatory motif (HRM), such as the bacterial iron response regulator protein.35 This work indicates that 1m has a role in heme catabolism. Heme oxygenase, first described by Tenhunen et al,36 is an intracellular enzyme that catalyzes the degradation of heme to biliverdin, CO, and free iron.37 Heme oxygenase is membrane-bound and found in microsomes and it may be speculated that its diverse functionsheme degradation, iron utilization, and production of the powerful antioxidant biliverdinare to be executed within the cells. 1m, on the other hand, is found extracellularly in most organs38-40 and is therefore expected to execute its tentative heme-degradation functions outside the cells. The presence of contaminations of heme oxygenase in the t-1m preparations would have explained their heme-degradative properties. However, no trace of heme oxygenase was found using commercial antibodies to the protein in Western blotting, suggesting that the effects indeed were caused by t-1m. Several heme-binding and antioxidative substances are present in plasma, for example, albumin, hemopexin, vitamin E, and ascorbic acid. It is therefore not likely that the heme-binding and degradation properties of 1m are primarily intended for free hemoglobin in plasma. More likely targets for these properties of 1m are extravascular ruptured erythrocyte membranes. The oxidized forms of precipitated hemoglobin, free heme, and iron found on the cytosolic face of erythrocyte membranes are highly toxic to neighboring tissue. A role of 1m could therefore be to protect against the exposed heme on the erythrocyte membranes, especially during extravascular hemolysis or massive intravascular hemolysis, where these plasma antiheme factors are not available in sufficient amounts. Both free monomeric 1m and high-molecular-weight 1m have been found widely distributed in extravascular compartments.38-40 IgA-1m has been isolated from the placenta.40 Interestingly, a truncated form of 1m with an apparent molecular mass around 30 kd was observed associated with a placenta membrane fraction. This 1m variant may be identical to t-1m, the activation product of erythrocyte membranes, described in this paper. Indeed, it can be speculated that an activation of free 1m and IgA-1m takes place in ruptured tissue cells, for instance during inflammation and necrosis. Instead of hemoglobin, however, which is confined to erythrocytes, 1m may interact with other heme-binding proteins such as cytochrome c in tissue cells. The t-1m found in urine may very well originate from extravascular tissue rather than blood, since the protein was shown to be rapidly transported between the 2 compartments.41 It remains to be shown, however, whether 1m can be processed and activated by all cells.     Acknowledgments Victoria Rydengård and Kerstin Nilsson are acknowledged for excellent technical performance. The authors are indebted to Dr Lars Hederstedt for generous help with the preparation of radiolabeled heme.     Footnotes Submitted July 11, 2001; accepted November 1, 2001. Supported by grants from the Swedish Medical Research Council (project no. 7144), EU-Biotech (project no. BIO4-CT98-0420), King Gustav V's 80-year foundation, the Swedish Society for Medical Research, the Royal Physiographic Society in Lund, Swedish Society of Medicine, and the Foundations of Greta and Johan Kock, Magnus Bergvall, and Crafoord. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Bo Åkerström, Department of Cell and Molecular Biology, B14, BMC, S-221 84 Lund, Sweden; e-mail: bo.akerstrom{at}medkem.lu.se.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Everse J, Hsia N. The toxicities of native and modified hemoglobins. Free Rad Biol Med. 1997;22:1075-1099[CrossRef][Medline] [Order article via Infotrieve]. 2. Faivre B, Menu P, Labrude P, Vigneron C. Hemoglobin autooxidation/oxidation mechanisms and methemoglobin prevention or reduction processes in the bloodstream. Art Cells Blood Subs Immob Biotech. 1998;26:17-26[Medline] [Order article via Infotrieve]. 3. Bunn HF. Hemoglobin. In: Haeberli A, ed. Human Protein Data, 1st Installment. Weinheim, Germany: VCH; 1992. 4. Winterbourn CC. Oxidative reactions of hemoglobin. Methods Enzymol. 1990;186:265-272[Medline] [Order article via Infotrieve]. 5. Sutton HC, Roberts PB, Winterbourn CC. The rate of reaction of superoxide radical with oxyhaemoglobin and methaemoglobin. Biochem J. 1976;155:503-510[Medline] [Order article via Infotrieve]. 6. Fiehn W. In: Zander R, Mertzlufft F, eds. Concentrations of methemoglobin in blood under physiological and pathological conditions. The Oxygen Status of Arterial Blood. Basel, Switzerland: Kaeger; 1991:190. 7. Browne P, Shalev O, Hebbel RP. The molecular pathobiology of cell membrane iron: the sickle red cell as a model. Free Rad Biol Med. 1998;24:1040-1048[CrossRef][Medline] [Order article via Infotrieve]. 8. Bunn HF, Forget BG. In: Hemoglobin. Mol, Gen and Clin Aspects. Philadelphia, PA: Saunders; 1986:634-662. 9. Chiancone E, Alfsen A, Ioppolo C, et al. Studies on the reaction of haptoglobin with haemoglobin and haemoglobin chains. I. Stoichiometry and affinity. J Mol Biol. 1968;34:347-356[CrossRef][Medline] [Order article via Infotrieve]. 10. Ekström B, Berggård I. Human 1-microglobulin: purification procedure, chemical and physicochemical properties. J Biol Chem. 1977;252:8048-8057[Free Full Text]. 11. Ekström B, Peterson PA, Berggård I. A urinary and plasma 1-glycoprotein of low molecular weight: isolation and some properties. Biochem Biophys Res Com. 1975;65:1427-1433[CrossRef][Medline] [Order article via Infotrieve]. 12. Tejler L, Grubb AO. A complex-forming glycoprotein heterogeneous in charge and present in human plasma, urine, and cerebrospinal fluid. Biochim Biophys Acta. 1976;439:82-94[Medline] [Order article via Infotrieve]. 13. Åkerström B, Lögdberg L. An intriguing member of the lipocalin protein family: 1-microglobulin. Trends Biochem Sci. 1990;15:240-243[CrossRef][Medline] [Order article via Infotrieve]. 14. Åkerström B, Lögdberg L, Berggård T, Osmark P, Lindqvist A. 1-microglobulina yellow-brown lipocalin. Biochim Biophys Acta. 2000;1482:172-184[CrossRef][Medline] [Order article via Infotrieve]. 15. Flower DR. The lipocalin protein family: structure and function. Biochem J. 1996;318:1-14. 16. Berggård T, Cohen A, Persson P, et al. 1-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound. Prot Sci. 1999;8:2611-2620[Medline] [Order article via Infotrieve]. 17. Grubb A, Méndez E, Fernández-Luna JL, Lopez C, Mihaesco E, Vaerman J-P. The molecular organization of the protein HC-IgA complex (HC-IgA). J Biol Chem. 1986;261:14313-14320[Abstract/Free Full Text]. 18. Åkerström B, Bratt T, Enghild JJ. Formation of the 1-microglobulin chromophore in mammalian and insect cells: a novel post-translational mechanism? FEBS Lett. 1995;362:50-54[CrossRef][Medline] [Order article via Infotrieve]. 19. Berggård T, Thelin N, Falkenberg C, Enghild JJ, Åkerström B. Prothrombin, albumin and immunoglobulin A form covalent complexes with 1-microglobulin in human plasma. Eur J Biochem. 1997;245:676-683[Medline] [Order article via Infotrieve]. 20. Elbashir MI, Nilson BHK, Åkesson P, Björck L, Åkerström B. Antibody response in immunized rabbits measured with bacterial immunoglobulin-binding proteins. J Immunol Meth. 1990;135:171-179[CrossRef][Medline] [Order article via Infotrieve]. 21. Babiker-Mohamed H, Olsson ML, Winquist O, Nilson BHK, Lögdberg L, Åkerström B. Characterization of monoclonal anti-1-microglobulin antibodies: binding strength, binding sites, and inhibition of 1-microglobulin-induced lymphocyte stimulation. Scand J Immunol. 1991;34:655-666[CrossRef][Medline] [Order article via Infotrieve]. 22. Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963;100:161-241. 23. Gattoni M, Boffi A, Sarti P, Chiancone E. Stability of the heme-globin linkage in ab dimers and isolated chains of human hemoglobin: a study of the heme transfer reaction from the immobilized proteins to albumin. J Biol Chem. 1996;271:10130-10136[Abstract/Free Full Text]. 24. Schiött T, Throne-Holst M, Hederstedt L. Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis. J Bacteriol. 1997;179:4523-4529[Abstract/Free Full Text]. 25. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685[CrossRef][Medline] [Order article via Infotrieve]. 26. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987;262:10035-10038[Abstract/Free Full Text]. 27. Wester L, Johansson MU, Åkerström B. Physicochemical and biochemical characterization of human 1-microglobulin expressed in baculovirus-infected insect cells. Protein Expr Purif. 1997;11:95-103[CrossRef][Medline] [Order article via Infotrieve]. 28. Greenwood FC, Hunter WM, Glover JS. The preparation of 131I-labelled human growth hormone. Biochem J. 1963;89:114-123[Medline] [Order article via Infotrieve]. 29. Villoutreix B, Åkerström B, Lindqvist A. Structural model of human 1-microglobulin: proposed scheme for the interaction with protein C. Blood Coagul Fibrinol. 2000;11:261-275[Medline] [Order article via Infotrieve]. 30. Amoresano A, Minchiotti L, Cosulich ME, et al. Structural characterization of the oligosaccharide chains of human 1-microglobulin from urine and amniotic fluid. Eur J Biochem. 2000;267:2105-2112[Medline] [Order article via Infotrieve]. 31. Peters T Jr. All about Albumin: Biochemistry, Genetics and Medical Applications. Orlando, FL: Academic Press; 1996. 32. Wester L, Fast J, Labuda T, et al. Carbohydrate groups of 1-microglobulin are important for secretion and tissue localization but not for immunological properties. Glycobiol. 2000;10:891-900[Abstract/Free Full Text]. 33. Lopez C, Grubb A, Mendez E. Human protein HC displays variability in its carboxyterminal amino acid sequence. FEBS Lett. 1982;144:349-353[CrossRef]. 34. Escribano J, Grubb A, Calero M, Méndez E. The protein HC chromophore is linked to the cysteine residue at position 34 of the polypeptide chain by a reduction-resistant bond and causes the charge heterogeneity of protein HC. J Biol Chem. 1991;266:15758-15763[Abstract/Free Full Text]. 35. Qi Z, Hamza I, O'Brian MR. Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Proc Natl Acad Sci U S A. 1999;96:13056-13061[Abstract/Free Full Text]. 36. Tenhunen R, Marver RS, Schmid J. Microsomal heme oxygenase: characterization of the enzyme. J Biol Chem. 1969;244:6388-6394[Abstract/Free Full Text]. 37. Yoshida T, Migita CT. Mechanism of heme degradation by heme oxygenase. J Inorg Biochem. 2000;82:33-41[CrossRef][Medline] [Order article via Infotrieve]. 38. Ødum L, Nielsen HW. Human protein HC (1-microglobulin) and inter-alpha-trypsin inhibitor in connective tissue. Histochem J. 1994;26:799-803[Medline] [Order article via Infotrieve]. 39. Berggård T, Oury TD, Thørgersen IB, Åkerström B, Enghild JJ. 1-microglobulin is found both in blood and in most tissues. J Histochem Cytochem. 1998;46:887-893[Abstract/Free Full Text]. 40. Berggård T, Enghild JJ, Badve S, Salafia CM, Lögdberg L, Åkerström B. Histologic distribution and biochemical properties of 1-microglobulin in human placenta. Am J Reprod Immunol. 1999;41:52-60. 41. Larsson J, Wingårdh K, Berggård T, et al. Distribution of 125I-labelled 1-microglobulin in rats after intravenous injection. J Lab Clin Med. 2001;137:165-175[CrossRef][Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: L. Andersson, B. Haraldsson, C. Johansson, and L. Barregard Methodological issues on the use of urinary alpha-1-microglobuline in epidemiological studies Nephrol. Dial. Transplant., April 1, 2008; 23(4): 1252 - 1256. [Abstract] [Full Text] [PDF] B. Akerstrom, G. J. Maghzal, C. C. Winterbourn, and A. J. Kettle The Lipocalin {alpha}1-Microglobulin Has Radical Scavenging Activity J. Biol. Chem., October 26, 2007; 282(43): 31493 - 31503. [Abstract] [Full Text] [PDF] S. Fagoonee, J. Gburek, E. Hirsch, S. Marro, S. K. Moestrup, J. M. Laurberg, E. I. Christensen, L. Silengo, F. Altruda, and E. Tolosano Plasma Protein Haptoglobin Modulates Renal Iron Loading Am. J. Pathol., April 1, 2005; 166(4): 973 - 983. [Abstract] [Full Text] [PDF] A. Sala, M. Campagnoli, E. Perani, A. Romano, S. Labo, E. Monzani, L. Minchiotti, and M. Galliano Human {alpha}-1-Microglobulin Is Covalently Bound to Kynurenine-derived Chromophores J. Biol. Chem., December 3, 2004; 279(49): 51033 - 51041. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Allhorn, M. Articles by Åkerström, B. Search for Related Content PubMed PubMed Citation Articles by Allhorn, M. Articles by Åkerström, B. Related Collections Red Cells Plenary Papers Social Bookmarking                   What's this?

	Copyright © 2002 by American Society of Hematology         Online ISSN: 1528-0020