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Hemopexin as an acute-phase protein binds heme with a high affinity and participates in a variety of processes of physiology and pathology. Hemopexin plays an important role in regulating inflammatory response by regulating pro-/anti-inflammatory cytokines. In this study, the hemopexin gene was identified from black rockfish. The open reading frame of the black rockfish hemopexin (SsHPX) gene was predicted and amplified. The different expression patterns of SsHPX in microbial-challenged tissues and peripheral blood leukocytes (PBLs) were analyzed by RT-qPCR. The results indicated that SsHPX showed high evolutionary conservations in sequence and structure. The expression of SsHPX was significantly induced in a time-dependent manner in black rockfish intestine after Edwardsiella piscicida infection, while the evidently down-regulations were observed in the PBLs following the stimulations of two microbial ligands. The recombinant SsHPX protein (rSsHPX) exhibited microbial binding ability. In addition, rSsHPX could reduce bacterial proliferation with effect, thereby alleviating the inflammatory responses induced by bacteria. Moreover, almost all the expressions of pro-inflammatory cytokines (tnfα, il-1β, il-8 and il-17c) and anti-inflammatory cytokine (IL-10) were inhibited in black rockfish intestine cells stimulated by the mixing solution of LPS + rSsHPX comparing with the group with LPS stimulation. Overall, this study provides a theoretical basis for investigating the key roles of an acute-phase protein hemopexin in bacteriostatic ability and inflammatory regulations in black rockfish during bacterial infection. These findings also contribute to a better understanding of the relatively stable and conserved biological function of hemopexin during species evolution.

Free heme released from hemoglobin contributes to exacerbated inflammation and tissue damage in hemolytic diseases. While a moderate level of free heme does not cause intravascular inflammation by itself, its presence during infection greatly enhances inflammation. Although specific serum proteins have been found to affect heme-induced inflammation, the selective contribution of serum proteins inhibiting macrophage activation by heme or, conversely, amplifying the production of cytokines by macrophages stimulated with heme and microbial molecules, is poorly defined. Here we identified a serum fraction containing proteins with >50 KDa which was capable of inhibiting heme-stimulated TNF production and capable of enabling TNF production under conditions of a heme-LPS synergy. The inhibition of heme-induced TNF production was mimicked by Hemopexin (Hx), human serum albumin (HSA), serum from Hx-knockout mice, and less efficiently by serum from albumin-knockout mice, but not by serum LDL. Hx and HSA inhibited heme-induced ROS generation, MAPK/ Syk phosphorylation and cell death. However, Hx and HSA each also promoted the synergistic relationship between heme and LPS upon TNF production. Serum from Hx-knockout mice was fully capable of enabling this synergy, while serum from albumin-knockout mice was less efficient to promote TNF production under these conditions. Low concentrations of HSA mimicked the ability of serum to enable heme-stimulated IL-1β production after LPS priming, while high concentrations inhibited it. Together, our findings indicate how heme inflammatory effects are restrained in the blood upon sterile hemolysis, yet exacerbate inflammation in the presence of microbes. Moreover, it is interesting to note that opposing effects of serum proteins on heme-induced macrophage activation were selected through evolution, with both effects exerted by Hx and albumin.

Age-related morbidity has become an increasingly significant issue worldwide. Sarcopenia, the decline in skeletal muscle mass and strength with age, has been reported to be a risk factor for cognitive impairment. Our previous study revealed that skeletal muscle atrophy shifts the onset of memory dysfunction earlier in young Alzheimer's disease mice and found that hemopexin is a myokine responsible for memory loss. This study aimed to elucidate the occurrence of memory impairment due to skeletal muscle atrophy in non-genetically engineered healthy young mice and the involvement of hemopexin. Closed-colony ddY mice at 12-13 weeks of age were used. Both hind limbs were immobilized by cast attachment for 14 d. Casting for 2 weeks induced a loss of skeletal muscle weight. The memory function of the mice was evaluated using a novel object recognition test. The cast-attached mice exhibited memory impairment. Hemopexin levels in the conditioned medium of the skeletal muscle, plasma, and hippocampus were increased in cast-attached mice. Continuous intracerebroventricular hemopexin infusion induced memory deficits in non-cast mice. To investigate whether hemopexin is the main causative factor of cognitive impairment, cast-attached mice were intracerebroventricularly infused with an anti-hemopexin antibody. Cast-induced memory impairment was reversed by the infusion of an anti-hemopexin antibody. These findings provide new evidence that skeletal muscle atrophy causes memory impairment in healthy young mice through the action of hemopexin in the brain., Competing Interests: Declaration of competing interest I have nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary: Physical exercise has profound effects on quality of life and susceptibility to chronic disease; however, the regulation of skeletal muscle function at the molecular level after exercise remains unclear. We tested the hypothesis that the benefits of exercise on muscle function are linked partly to microtraumatic events that result in accumulation of circulating heme. Effective metabolism of heme is controlled by Heme Oxygenase-1 (HO-1, Hmox1), and we find that mouse skeletal muscle-specific HO-1 deletion (Tam-Cre-HSA-Hmox1fl/fl) shifts the proportion of muscle fibers from type IIA to type IIB concomitant with a disruption in mitochondrial content and function. In addition to a significant impairment in running performance and response to exercise training, Tam-Cre-HSA-Hmox1fl/fl mice show remarkable muscle atrophy compared to Hmox1fl/fl controls. Collectively, these data define a role for heme and HO-1 as central regulators in the physiologic response of skeletal muscle to exercise.

We previously reported that the highly reactive cell-free heme (CFH) is increased in the plasma of patients with chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time that CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl2, with plasmalogens) are increased in the plasma of patients exposed to Cl2 gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloride-sensitive epithelial Na+ channel (ENaC) and cation sodium (Na+) channels in mouse alveolar cells and trans-epithelial Na+ transport across human airway cells with EC50 of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86–1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na+ transport across the channel. A single intramuscular injection of the heme-scavenging protein, hemopexin (4 μg/kg body weight), one hour post halogen gas exposure, decreased plasma CFH and improved lung ENaC activity in mice. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.

Summary: Hemopexin (Hx) is a scavenger of labile heme. Herein, we present data defining the role of tumor stroma-expressed Hx in suppressing cancer progression. Labile heme and Hx levels are inversely correlated in the plasma of patients with prostate cancer (PCa). Further, low expression of Hx in PCa biopsies characterizes poorly differentiated tumors and correlates with earlier time to relapse. Significantly, heme promotes tumor growth and metastases in an orthotopic murine model of PCa, with the most aggressive phenotype detected in mice lacking Hx. Mechanistically, labile heme accumulates in the nucleus and modulates specific gene expression via interacting with guanine quadruplex (G4) DNA structures to promote PCa growth. We identify c-MYC as a heme:G4-regulated gene and a major player in heme-driven cancer progression.Collectively, these results reveal that sequestration of labile heme by Hx may block heme-driven tumor growth and metastases, suggesting a potential strategy to prevent and/or arrest cancer dissemination.

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

Proinflammatory cytokines, including TNF-α and IL-6, can contribute to insulin resistance. Conversely, insulin has some actions that can be considered anti-inflammatory. Hemopexin is a Class 2 acute phase reactant and control of its transcription is predominantly regulated by IL-6, with TNF-α and IL-1β also inducing hemopexin gene expression. Thus, we asked whether insulin could inhibit the ability of TNF-α to stimulate hemopexin mRNA expression. In cultured rat hepatoma (H4IIE) cells, TNF-α significantly increased hemopexin mRNA accumulation. The TNF-α-induced increase of hemopexin mRNA was dramatically attenuated by insulin, even though TNF-α reduced peak insulin activation of ERK. Thus, even though TNF-α can contribute to insulin resistance, the residual insulin response was still able to counteract TNF-α actions.

Background: Approximately 180 mothers are treated in Swedish Intensive Care Units (ICU) due to preeclampsia each year. Although several clinical risk factors are known, prediction of critical disease is challenging. Two scavenger proteins, hemopexin (Hpx) and alpha-1-microglobulin (A1M) have been suggested to be associated with the oxidative stress seen in preeclampsia. The ratio of two other biomarkers, soluble fms-like tyrosine kinase (sFlt-1) to placental growth factor (PIGF), is predictive of adverse pregnancy outcomes., Methods: In total 121 women were included in this study where we compared risk factors for preeclampsia, plasma levels of Hpx and A1M in ICU-patients with preeclampsia (n = 41) compared to uncomplicated preeclampsia cases (n = 40) and normotensive pregnancies (n = 40), with the objective to identify clinical risk patterns for severe disease. The sFlt-1/PIGF ratio was investigated in early and late onset preeclampsia ICU-patients. Blood samples were collected at admission to ICU and within 27 h postpartum for all groups., Results: Hemopexin and A1M levels were significantly lower in the preeclampsia ICU-cohort compared to uncomplicated preeclampsia patients. The sFlt-1/PIGF-ratio was elevated in the ICU-patients but there was no difference between early and late onset preeclampsia. The ICU-patients had more clinical risk factors, refractory hypertension, and an increased rate of emergency Caesarean section., Conclusion: Intensive care patients have more clinical risk factors and a Hpx and A1M profile suggestive of depletion and thereby a reduced capacity to respond to oxidative stress. The ratios of sFlt-1/PIGF were high in the ICU-cohort and in accordance with pre-delivery levels predictive of adverse pregnancy outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hemopexin, a heme scavenging protein, accumulates in the kidneys during acute kidney injury (AKI). However, the function of this accumulated hemopexin in the kidney is unclear. In both the cisplatin-induced and the unilateral kidney ischemia-reperfusion injury models of AKI, we found accumulation of hemoglobin and hemopexin in the kidneys localized to the proximal tubules. Next, hemopexin wild-type and knockout mice were compared in both AKI models and hemopexin wild type mice had significantly worse kidney injury. Furthermore, there was increased kidney expression of kidney injury molecule-1 (a biomarker of AKI) and heme oxygenase-1 (an indicator of oxidative stress) in hemopexin wild type compared with knockout mice in both models of AKI. Next, the interaction of hemopexin and hemoglobin in vitro was investigated using cultured proximal tubular cells. Co-incubation of hemopexin with hemoglobin resulted in hemoglobin deposition and exaggerated hemoglobin-induced injury. Deferoxamine, an iron chelator, and ferrostatin-1, a ferroptosis inhibitor, inhibited this deleterious effect of hemoglobin and hemopexin in proximal tubular cells, implicating iron toxicity in the mechanism of hemopexin mediated injury. Furthermore, the protective effect of deferoxamine in cisplatin-induced AKI was apparent in hemopexin wild type, but not in hemopexin knockout mice, further implicating hemopexin as a mediator of iron toxicity in AKI. Thus, our findings demonstrate that hemopexin accumulates in the kidneys and worsens kidney injury in AKI by increasing hemoglobin deposition on proximal tubular cells to exaggerate hemoglobin-induced cell injury., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

During hemolysis, erythrophagocytes dispose damaged red blood cells. This prevents the extracellular release of hemoglobin, detoxifies heme, and recycles iron in a linked metabolic pathway. Complementary to this process, haptoglobin and hemopexin scavenge and shuttle the red blood cell toxins hemoglobin and heme to cellular clearance. Pathological hemolysis outpaces macrophage capacity and scavenger synthesis across a diversity of diseases. This imbalance leads to hemoglobin-driven disease progression. To meet a void in treatment options, scavenger protein-based therapeutics are in clinical development., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Research Question: Do pregnancies with corpus luteum show different maternal and fetal plasma concentrations of the scavenger proteins haemopexin and α 1 -microglobulin compared with pregnancies without corpus luteum in preeclampsia?, Design: Case-control study of 160 singleton pregnancies: 54 naturally conceived, 50 by IVF after fresh embryo transfer or frozen embryo transfer (FET) in natural cycle (presence of corpus luteum) and 56 after fresh oocyte donation or FET in programmed cycles (absence of corpus luteum). Pregnancies were subclassified into normotensive, preeclampsia and severe preeclampsia cases. Heme-scavenger concentrations were measured by ELISA in maternal and cord plasma collected at delivery., Results: After adjustment, maternal haemopexin was higher in IVF with corpus luteum than in naturally conceived pregnancies in normotensive (P = 0.038) and preeclampsia (P = 0.011) populations, and lower in preeclampsia for IVF pregnancies lacking corpus luteum compared with IVF with corpus luteum (P = 0.002). Maternal α 1 -microglobulin levels were higher in the absence of corpus luteum only in severe cases of preeclampsia compared with naturally conceived pregnancies (P = 0.014) and IVF with corpus luteum pregnancies (P = 0.041). In cord blood, haemopexin was higher in IVF with corpus luteum compared with naturally conceived pregnancies in preeclampsia (P = 0.039) and α 1 -microglobulin was higher in the group lacking corpus luteum compared with IVF with corpus luteum in the normotensive population (P < 0.001)., Conclusions: The physiological differences shown for these heme-scavengers between pregnancies after embryo transfer in the presence or absence of corpus luteum support the hypothesis that corpus luteum activity could influence perinatal outcomes. Future research is needed on whether applying potential strategies to develop a corpus luteum might reduce the perinatal complications associated with programmed cycles of IVF., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Thrombotic microangiopathy, hemolysis and acute kidney injury are typical clinical characteristics of hemolytic-uremic syndrome (HUS), which is predominantly caused by Shiga-toxin-producing Escherichia coli. Free heme aggravates organ damage in life-threatening infections, even with a low degree of systemic hemolysis. Therefore, we hypothesized that the presence of the hemoglobin- and the heme-scavenging proteins, haptoglobin and hemopexin, respectively impacts outcome and kidney pathology in HUS. Here, we investigated the effect of haptoglobin and hemopexin deficiency (haptoglobin -/- , hemopexin -/- ) and haptoglobin treatment in a murine model of HUS-like disease. Seven-day survival was decreased in haptoglobin -/- (25%) compared to wild type mice (71.4%), whereas all hemopexin -/- mice survived. Shiga-toxin-challenged hemopexin -/- mice showed decreased kidney inflammation and attenuated thrombotic microangiopathy, indicated by reduced neutrophil recruitment and platelet deposition. These observations were associated with supranormal haptoglobin plasma levels in hemopexin -/- mice. Low dose haptoglobin administration to Shiga-toxin-challenged wild type mice attenuated kidney platelet deposition and neutrophil recruitment, suggesting that haptoglobin at least partially contributes to the beneficial effects. Surrogate parameters of hemolysis were elevated in Shiga-toxin-challenged wild type and haptoglobin -/- mice, while signs for hepatic hemoglobin degradation like heme oxygenase-1, ferritin and CD163 expression were only increased in Shiga-toxin-challenged wild type mice. In line with this observation, haptoglobin -/- mice displayed tubular iron deposition as an indicator for kidney hemoglobin degradation. Thus, haptoglobin and hemopexin deficiency plays divergent roles in Shiga-toxin-mediated HUS, suggesting haptoglobin is involved and hemopexin is redundant for the resolution of HUS pathology., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Hemopexin, a high affinity heme-binding protein is widely involved in variety physiological and pathological processes. It is an important acute phase response protein, and is important in regulating the inflammatory response. In this study, the open reading frame of Nile tilapia hemopexin (OnHpx) gene was amplified. The expression pattern of OnHpx in natural and bacterial challenged tilapia tissues were analyzed through RT-qPCR. The results indicated the OnHpx was most abundant in liver, and increased significantly in liver, spleen, head kidney and peripheral blood after bacterial challenge. Furthermore, the OnHpx mRNA was also significantly up-regulated in monocytes/macrophages and hepatocytes under the stimulation of S. agalactiae or A. hydrophila. In addition, the recombinant OnHpx protein could effectively reduce the bacteria proliferation and alleviate the inflammatory reaction caused by bacteria. Moreover, the (r)OnHpx also regulated the respiratory burst of monocytes/macrophages and played an important role in the antioxidant process. To our knowledge, these results provide the first evidence on the antibacterial and anti-inflammatory response mechanism of Hpx in early vertebrates. This brings new insights about the understanding of the evolutionary origins and ancient roles of the Hpx in the innate immune defense., (Copyright © 2021. Published by Elsevier B.V.)

Rhabdomyolysis is a life-threatening condition caused by skeletal muscle damage with acute kidney injury being the main complication dramatically worsening the prognosis. Specific treatment for rhabdomyolysis-induced acute kidney injury is lacking and the mechanisms of the injury are unclear. To clarify this, we studied intra-kidney complement activation (C3d and C5b-9 deposits) in tubules and vessels of patients and mice with rhabdomyolysis-induced acute kidney injury. The lectin complement pathway was found to be activated in the kidney, likely via an abnormal pattern of Fut2-dependent cell fucosylation, recognized by the pattern recognition molecule collectin-11 and this proceeded in a C4-independent, bypass manner. Concomitantly, myoglobin-derived heme activated the alternative pathway. Complement deposition and acute kidney injury were attenuated by pre-treatment with the heme scavenger hemopexin. This indicates that complement was activated in a unique double-trigger mechanism, via the alternative and lectin pathways. The direct pathological role of complement was demonstrated by the preservation of kidney function in C3 knockout mice after the induction of rhabdomyolysis. The transcriptomic signature for rhabdomyolysis-induced acute kidney injury included a strong inflammatory and apoptotic component, which were C3/complement-dependent, as they were normalized in C3 knockout mice. The intra-kidney macrophage population expressed a complement-sensitive phenotype, overexpressing CD11b and C5aR1. Thus, our results demonstrate a direct pathological role of heme and complement in rhabdomyolysis-induced acute kidney injury. Hence, heme scavenging and complement inhibition represent promising therapeutic strategies., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: High blood cholesterol is associated with atherogenesis and endothelial dysfunction. The latter is present in hemolytic diseases, such as sickle cell anemia, whose carriers have hypocholesterolemia and low incidence of coronary artery disease., Objective: We aimed to characterize cholesterol fractions in patients with sickle cell disease and explore the relationship among lipoproteins, varying degrees of hemolysis, and its biomarkers., Methods: We recruited 37 healthy individuals, 39 with hemoglobin SC disease, and 40 with sickle cell anemia and quantified cholesterol fractions, heme resulting from hemoglobin breakdown, and its main scavenger protein hemopexin., Results: Hypocholesterolemia was most significant in patients with sickle cell anemia, and cholesterol levels correlated positively with hemopexin. Nevertheless, patients still had higher relative low-density lipoprotein (LDL) oxidation than healthy subjects. Analysis of lipoproteins isolated by density ultracentrifugation showed that the LDL fraction contained higher concentrations of heme than the high-density lipoprotein (HDL) fraction, whereas HDL contained more hemopexin than LDL, albeit greatly reduced in patients., Conclusion: Our findings show that the abnormally low lipoprotein levels in sickle cell anemia correlate with hemolysis markers, particularly with hemopexin concentrations, along with significant reduction of this heme scavenger in HDL fractions. This may suggest an important role for HDL in the defense against heme-induced endothelial dysfunction in hemolytic diseases., (Copyright © 2018 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alternations in the glycosylation of proteins have been described in connection with several cancers, including hepatocellular carcinoma (HCC) and colorectal cancer. Analytical tools, which use combination of liquid chromatography and mass spectrometry, allow precise and sensitive description of these changes. In this study, we use MRM and FT-ICR operating in full-MS scan, to determine ratios of intensities of specific glycopeptides in HCC, colorectal cancer, and liver metastasis of colorectal cancer. Haptoglobin, hemopexin and complement factor H were detected after albumin depletion and the N-linked glycopeptides with fucosylated glycans were compared with their non-fucosylated forms. In addition, sialylated forms of an O-linked glycopeptide of hemopexin were quantified in the same samples. We observe significant increase in fucosylation of all three proteins and increase in bi-sialylated O-glycopeptide of hemopexin in HCC of hepatitis C viral (HCV) etiology by both LC-MS methods. The results of the MRM and full-MS scan FT-ICR analyses provide comparable quantitative readouts in spite of chromatographic, mass spectrometric and data analysis differences. Our results suggest that both workflows allow adequate relative quantification of glycopeptides and suggest that HCC of HCV etiology differs in glycosylation from colorectal cancer and liver metastasis of colorectal cancer., Significance: The article compares N- and O-glycosylation of several serum proteins in different diseases by a fast and easy sample preparation procedure in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry. The results show successful glycopeptides relative quantification in a complex peptide mixture by the high resolution instrument and the detection of glycan differences between the different types of cancer diseases. The presented method is comparable to conventional targeted MRM approach but allows additional curation of the data., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Serum high and low density lipoproteins, albumin, and hemopexin (HDL, LDL, SA, and HPX, respectively) serve as traps of toxic plasma heme and participate in its complete clearance by transportation to the liver. Moreover, SA-(heme) and HPX-heme have been proposed to facilitate NO scavenging in vivo. Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins (HDL-heme-NO and LDL-heme-NO, respectively) as well as of ferrous nitrosylated heme-rabbit serum hemopexin (HPX-heme-NO) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO). HDL-heme-NO and LDL-heme-NO as well as SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are five-coordinate heme-iron species, characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum. On the other hand, SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO are six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry. The heme-iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-heme-NO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL>HDL>SA>HPX. Altogether, these observations suggest that HPX displays a cleft much more suitable for heme binding than other heme-carriers.

An immunoturbidometric assay was developed for the measurement of proteins in culture fluids of hepatocytes. The assay is simple to perform and avoids the biohazards associated with radioimmunoassays. The limit of detection of this assay exemplified by hemopexin and transferrin is 5 ng/ml protein. This degree of sensitivity is attained by incorporating into the procedure the addition of polyethylene glycol to enhance formation of primary antigen-antibody complexes and of a second antibody to further increase the immune complex size, which favors the ratio of specific to background light scattering.