Porcine Hemoglobin Research Articles

Non-Specific Binding and Cross-Reaction of ELISA: A Case Study of Porcine Hemoglobin Detection.

Different types of enzyme-linked immunosorbent assays (ELISA) have been widely used to control food safety and quality. To develop an accurate and reproducible ELISA, false immunodetection results caused by non-specific binding (NSB) and cross-reaction must be prevented. During the case study of sandwich ELISA development for the detection of porcine hemoglobin (PHb), several critical factors leading to NSB and cross-reaction were found. First, to reduce the NSB of the target analyte, the selection of microplate and blocker was discussed. Second, cross-reactions between enzyme-labeled secondary antibodies and sample proteins were demonstrated. In addition, the function of (3-aminopropyl)triethoxysilane (APTES) was evaluated. Overall, this study highlights the essence of both antibody and assay validation to minimize any false-positive/negative immunodetection results.

Ferrous hemoglobin and hemoglobin-based oxygen carriers acting as a peroxidase can inhibit oxidative damage to endothelial cells caused by hydrogen peroxide.

Oxidative damage caused by the ferryl hemoglobin is one of the major clinical adverse reactions of hemoglobin-based oxygen carriers (HBOCs), while the production of reactive oxygen species in a pathological state can oxidize hemoglobin (HbFe2+ ) to ferryl Hb, which can then enter the pseudoperoxidase cycle, making hemoglobin highly toxic. In this study, we found that ferrous hemoglobin and polymerized porcine hemoglobin (one of the HBOCs) have the peroxidase activity different from the pseudoperoxidase activity of ferric hemoglobin. Ferrous hemoglobin can catalyze the reaction of tyrosine (Tyr) with hydrogen peroxide. In addition, the results also indicated that ferrous hemoglobin and pPolyHb have a strong inhibitory effect on the pseudoperoxidase activity of ferric hemoglobin. Therefore, hydrogen peroxide was consumed in a large amount, which greatly prevented hemoglobin from becoming oxidized and entering the pseudoperoxidase cycle, thus inhibiting ferryl Hb toxicity. We further cultured human umbilical vein endothelial cells and monitored cell morphology, viability, cell cycle, apoptosis, lactate dehydrogenase (LDH) release, and malondialdehydes (MDAs) formation when incubated with H2 O2 , Tyr, and HbFe2+ . HbFe2+ and pPolyHb reduced cell cycle arrest, apoptosis, LDH release, and MDA formation. These results showed that reducing oxidative damage induced by H2 O2 and converted hemoglobin from a molecule that is toxic to one that inhibits oxidative damage, suggesting a new strategy for development of a safer HBOCs.

Interactions of Gallic Acid with Porcine Hemoglobin: Effect on the Redox State and Structure of Hemoglobin.

The effect of gallic acid (GA) on the redox state of hemoglobin (Hb) and the structural mechanism upon the Hb-GA interaction were investigated. Results indicated that GA exhibited antioxidant and pro-oxidant effects on Hb, which depended on its concentration and the redox state of Hb. The antioxidant capacity of GA contributed to the inhibition of free iron release from Hb. GA could bind to the central cavity of Hb and interacted with the heme moiety through direct hydrophobic contacts as indicated by docking analysis, but GA did not disrupt the heme structure. Conversely, GA increased the compactness of the Hb molecule and might narrow the crevice around the heme pocket, which contributed to the inhibition of Hb autoxidation and the free iron release. Results provided significant insights into the interaction of GA with redox-active Hb, which is beneficial to the application of GA in relative meat and blood products.

Production of Taste Enhancers from Protein Hydrolysates of Porcine Hemoglobin and Meat Using Bacillus amyloliquefaciens γ-Glutamyltranspeptidase.

To improve the flavor of hydrolysates from porcine hemoglobin and meat, γ-glutamyltranspeptidase (GGT) from Bacillus amyloliquefaciens was added to catalyze the formation of kokumi γ-glutamyl peptides via a γ-glutamyl transfer reaction. Quantitation of free amino acids and γ-glutamyl dipeptides was carried out in combination with sensory analysis. Sensory perception, especially the thick, complex, continuous, and overall kokumi sensation of both hemoglobin and meat hydrolysates, was greatly enhanced by γ-glutamylation. Due to the higher amount of glutamine present in meat hydrolysates, γ-glutamylated hydrolysates from meat contained higher concentrations of γ-glutamyl dipeptides and showed stronger kokumi sensation than the hemoglobin counterpart without the addition of glutamine. For hydrolysates from both raw materials, extra addition of glutamine (10 and 20 mM) was beneficial for obtaining higher concentrations of γ-glutamyl dipeptides but contributed little to the kokumi sensation. This study revealed that the kokumi sensation of protein hydrolysates could be intensified by a γ-glutamyl transfer reaction, and the enhanced kokumi sensation could be related to the generation of γ-glutamyl peptides.

Temperature Self-Compensation Biosensor Based on LPG Concatenated With SNCS Structure

A biosensor based on long period grating (LPG) concatenated with single-mode-no-core-single-mode (SNCS) fiber structure is designed and demonstrated both theoretically and experimentally. By analyzing the opposite shift of transmission dips formed by LPG and SNCS, the RI and temperature variations can be demodulated to realize the temperature self-compensation. The RI sensitivity of 90.56 nm/RIU is obtained by experiment at 1.333-1.399 with temperature compensation coefficient of -1.33× 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-4</sup> /°C. The biosensing performance of the sensor is evaluated by detecting porcine hemoglobin (Hb) solutions with different concentrations and the sensitivity of 0.0165 nm/mg/mL is obtained. The designed sensor with simple structure, high sensitivity, noninvasive and label-free detection capability shows great potential in biosensing applications.

Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for porcine hemoglobin quantification

The major objective of this study was to establish a monoclonal antibody (mAb)-based sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of porcine hemoglobin (PHb) in raw meat products. Before assay development, two mAbs immunoreactive to PHb β subunit with different epitopes were characterized. The optimized immunoassay was specific to PHb and had a wide PHb working range from 15.6 µg/mL to 3,000 µg/mL and high reproducibility with low coefficient of variations (CV < 20%). Through this assay, the estimated PHb residuals in pork loin and shoulder meats were 0.4 mg/g and 1.1 mg/g, respectively. In addition, this immunoassay could effectively quantify PHb in laboratory-spiked meats (pork loin, pork shoulder, and turkey breast) with acceptable recovery. Overall, this is the first mAb-based sandwich ELISA that is suitable for the government, food industry, and third-party authority to monitor PHb residuals or porcine blood adulteration in raw pork and pork-free meat products.

Plastein from hydrolysates of porcine hemoglobin and meat using Alcalase and papain

Plastein is defined as a protease-induced peptide aggregate and has been explored for over a century. This study investigated the effects of Alcalase and papain on plastein formation in protein hydrolysates of porcine hemoglobin and meat by measuring turbidity, particle size distribution, free amino groups and chemical interactions, as well as identifying the soluble peptides remaining in solution by LC–MS/MS. The results showed that Alcalase induced more peptide aggregation than papain in terms of increases in turbidity and particle size. Porcine hemoglobin was better than meat in inducing plastein formation in a short reaction time. Besides, covalent bonds involving peptide bonds and disulfide bonds were not crucial in the plastein reaction, instead a high proportion of hydrophobic interactions dominated the plastein. Not all peptides of both hydrolysates took part in plastein formation, and the regions of sequence that were prone to aggregation were visualized by Peptigram.

Human Hemoglobin Subunit Beta Functions as a Pleiotropic Regulator of RIG-I/MDA5-Mediated Antiviral Innate Immune Responses.

Hemoglobin is an important oxygen-carrying protein and plays crucial roles in establishing host resistance against pathogens and in regulating innate immune responses. The hemoglobin subunit beta (HB) is an essential component of hemoglobin, and we have previously demonstrated that the antiviral role of the porcine HB (pHB) is mediated by promoting type I interferon pathways. Thus, considering the high homology between human HB (hHB) and pHB, we hypothesized that hHB also plays an important role in the antiviral innate immunity. In this study, we characterized hHB as a regulatory factor for the replication of RNA viruses by differentially regulating the RIG-I- and MDA5-mediated antiviral signaling pathways. Furthermore, we showed that hHB directly inhibited MDA5-mediated signaling by reducing the MDA5-double-stranded RNA (dsRNA) interaction. Additionally, hHB required hHB-induced reactive oxygen species (ROS) to promote RIG-I-mediated signaling through enhancement of K63-linked RIG-I ubiquitination. Taken together, our findings suggest that hHB is a pleiotropic regulator of RIG-I/MDA5-mediated antiviral responses and further highlight the importance of the intercellular microenvironment, including the redox state, in regulating antiviral innate immune responses.IMPORTANCE Hemoglobin, the most important oxygen-carrying protein, is involved in the regulation of innate immune responses. We have previously reported that the porcine hemoglobin subunit beta (HB) exerts antiviral activity through regulation of type I interferon production. However, the antiviral activities and the underlying mechanisms of HBs originating from other animals have been poorly understood. Here, we identified human HB (hHB) as a pleiotropic regulator of the replication of RNA viruses through regulation of RIG-I/MDA5-mediated signaling pathways. hHB enhances RIG-I-mediated antiviral responses by promoting RIG-I ubiquitination depending on the hHB-induced reactive oxygen species (ROS), while it blocks MDA5-mediated antiviral signaling by suppressing the MDA5-dsRNA interaction. Our results contribute to an understanding of the crucial roles of hHB in the regulation of the RIG-I/MDA5-mediated signaling pathways. We also provide novel insight into the correlation of the intercellular redox state with the regulation of antiviral innate immunity.

Effect of Tea Polyphenols on the Oxidation and Color Stability of Porcine Hemoglobin.

The oxidation and color stability of porcine hemoglobin (Hb) in the presence of tea polyphenols (TP), as well as the mechanism, were investigated using the methods of color and oxidation analyses, ultraviolet-visible and fluorescence spectroscopy. Results indicated that TP interacted with the tryptophan and tyrosine residues of Hb through inserting into its hydrophobic pocket. This interaction showed a concentration-dependent effect on Hb, which might lead to completely opposite results. The presence of TP (16 mg/L) disrupted Hb (16 mg/L) structure, and the exposure of heme iron facilitated the oxidation and discoloration of Hb. However, a lower level of TP should not break Hb structure but could work as an antioxidant and restrain the formation of methemoglobin. Consequently, TP (1.6 mg/L) considerably maintained the redness of Hb (16 mg/L, P < 0.05) when stored at pH 7.4 and 25 °C for 72 hr. Results may provide scientific information for the proper use of TP in blood and meat products. PRACTICAL APPLICATION: Proper utilization of tea polyphenols (TP) in food products is beneficial to improve antioxidant capacity and nutrition quality of food. We proved that it was potential to corporate TP into blood and meat products to prevent discoloration and oxidative deterioration.

PPolyHb protects myocardial H9C2 cells against ischemia-reperfusion injury by regulating the Pink1-Parkin-mediated mitochondrial autophagy pathway

Mitochondrial dysfunction is a major contributory factor for myocardial ischemia-reperfusion (I/R) injury. It has been reported that Pink1-Parkin-mediated mitochondrial autophagy could effectively remove damaged mitochondria and excess ROS to ensure the stability of intracellular mitochondria. The present study was designed to evaluate whether the polymerized porcine haemoglobin (pPolyHb), a novel type of haemoglobin oxygen carrier, has an effect on I/R injury via regulating the Pink1-Parkin mediated mitochondrial autophagy pathway in myocardial H9C2 cells. The results revealed that pPolyHb could effectively reduce apoptosis and improve the survival rates of H9C2 cells. In addition, Pink1 and Parkin levels gradually decreased with pPolyHb reoxygenation. The inhibition of mitochondrial autophagy through mitochondrial-division inhibitor-1(mdivi-1) resulted in a decrease in anti-apoptotic protein Bcl-2 and an increase in pro-apoptotic protein Bax and CytC. In conclusion, pPolyHb has a protective effect on myocardial ischemia-reperfusion injury by regulating moderate mitochondrial autophagy.

Protein hydrolysates of porcine hemoglobin and blood: Peptide characteristics in relation to taste attributes and formation of volatile compounds.

The objective of this study was to investigate the impact of endo- and exo-peptidase treatment on certain structural characteristics of peptides and volatile compounds of porcine hemoglobin and whole blood hydrolysates. Porcine hemoglobin and whole blood were hydrolyzed by endo- and exo-peptidases. The presence of exopeptidases reduced the bitterness and altered the volatile profiles of protein hydrolysates. Exopeptidase treatment can release terminal amino acids from peptides, which in turn may contribute to formation of volatile compounds by Maillard reactions. In contrast, endopeptidases conferred a slightly bitter taste and different volatile profiles. For hemoglobin hydrolysates, principal component analysis revealed that proteases were categorized into three groups based on endo- or exo-peptidase activity. Whole blood is a more complex raw material, yet the proteases were still categorized in a similar fashion. This work contributes to understanding structural characteristics responsible for taste and volatile profiles of protein hydrolysates.

Storage and thermal stability of novel heme‐based pigments prepared from porcine hemoglobin

AbstractThe stability of novel meat pigments derived from heme‐enriched extract obtained from porcine hemoglobin was assessed. Four different ligands (sodium nitrite, 4‐methylimidazole, methyl nicotinate, and pyrazine) were used to produce spray‐dried, microencapsulated heme‐ligand complexes. The storage and thermal stabilities of the produced pigments were assessed, over a 6‐months storage period and across a temperature range of 25–75°C. Color measurements and evaluation were made on the dissolved pigments by ultraviolet–visible absorbance spectroscopy and CIELAB color space. All heme‐ligand complexes exhibited a stable red color across the storage period, except for the heme‐methyl nicotinate adduct, which color faded to brown after 30 days of storage. For thermal stability, only the heme‐4‐methylimidazole complex did not retain its red color beyond 55°C. The redness of the heme‐pyrazine complex showed improvement upon heating, which is proposed to be due to the degradation of polymeric heme‐pyrazine structure formed during the ligation process.Practical applicationsDue to the global effort to reduce nitrite addition in food product, there is an important interest to replace it in processed meat products, wholly or in part. Additionally, the perspective of optimizing the usage of an under‐utilized blood fraction is attractive for the meat industry. The development of a heme pigment derived from porcine blood thus presents a good commercial potential. Two important aspects of such product would be its stability upon storage, or during heat treatment to levels similar to what is used in processed, cooked meat products. This study presents the behavior for those two aspects of different heme‐ligand complexes, and compares the results obtained with a heme pigment produced from the traditionally used nitrite.

Maltose-mediated long-term stabilization of freeze- and spray- dried forms of bovine and porcine hemoglobin

Slaughterhouse blood represents a valuable source of hemoglobin, which can be used in the production of heme-iron based supplements for the prevention/treatment of iron-deficiency anemia. In order to obtain a stable solid-state formulation, the effect of maltose addition (30 %) on the stability and storage of bovine and porcine hemoglobin in powders obtained by spray and freeze-drying (without maltose: Hb; with maltose: HbM) were investigated. Differential scanning calorimetry of spray- and freeze-dried powders indicated satisfying quality of the formulation prepared with maltose on dissolving back into solution. After two-year storage at room temperature (20?5?C) in solid forms, protected from moisture and light, rehydrated spray- and freeze-dried HbM were red, while Hb were brown. Dynamic light scattering showed the presence of native hemoglobin monomers in rehydrated spray- and freeze-dried HbM, but their agglomerates in Hb samples. UV?Vis spectrophotometry confirmed an absence of significant hemoglobin denaturation and methemoglobin formation in HbM freeze-dried powders. In spray-dried HbM, an increased level of methemoglobin was detected. The results confirmed the stabilizing effect of maltose, and suggested its use in the production of long-term stable solid-state formulations of hemoglobin, along with drying processes optimization.

Investigation of nitrite alternatives for the color stabilization of heme-iron hydrolysates.

This study investigates the potential of novel heme-ligand complexes, derived from heme-iron isolated from porcine hemoglobin by enzymatic hydrolysis, to use as pigments for meat products. Five alternatives to sodium nitrite were identified as possible heme ligands and stabilizing agents of the red conformation of heme. The effects of 4-methylimidazole, methyl nicotinate, pyrrolidine, piperidine, pyrazine and sodium nitrite (as comparative benchmark) on the color of heme-iron extract and pure hemin standard were studied in solution. The ligand affinity and heme-ligand stability was assessed over time in solution by UV-Vis absorbance spectroscopy and CIELAB color space parameters. The CIE redness score a* was used as a single measurement to propose a predictive model based on the following parameters: heme source (heme-iron extract or hemin standard), heme-to-ligand molar ratio (1:20 to 1:300), and storage time (up to 32days). The optimal concentration at which each ligand can be added to either heme source, as well as the stability of the red color of the formed heme-ligand complexes in-solution was determined. Heme-iron extract-derived samples showed increased redness and color stability as compared to their hemin counterparts. No ligand showed as much affinity for heme as sodium nitrite. As the most promising ligand candidates, methyl nicotinate and 4-methylimidazole started to show color changes at a 1:50 molar ratio, but higher amounts (1:100 and 1:300, respectively) were required to attain the maximum redness possible with the highest stability.

Study of the pharmacokinetics of polymerized porcine hemoglobin (pPolyHb)

Polymerized porcine hemoglobin (pPolyHb), a kind of glutaraldehyde-polymerized haemoglobin-based oxygen carrier, was developed as a potential red blood substitute for clinical applications. Assessment of its absorption, distribution, and metabolism is a major determinant of its safety and efficacy. In this study, a series of pharmacokinetic parameters of pPolyHb were measured and calculated in different animal models, such as the top-load model, the 50% exchange transfusion model and the haemorrhagic shock model. The results showed that the kinetic process of pPolyHb in rats conforms to the laws of linear pharmacokinetics in vivo. The half-life of pPolyHb was superior and more stable under non-pathological conditions, but in clinical situations such as trauma and anaemia, the half-life of pPolyHb may decline. The study of clearance (CL) and the apparent volume of distribution (Vd) of pPolyHb in these three different animal models demonstrated longer residence and a predominant, targeted role of pPolyHb with little accumulation in tissues. Through this study, the superior pharmacokinetic characteristics of pPolyHb have been proved and will aid in the determination of a reasonable dosing regimen and administration interval in clinical situations.

Development of Volatile Compounds during Hydrolysis of Porcine Hemoglobin with Papain.

There is a growing market for the use of hydrolysates from animal side-streams for production of high-protein supplements. However, there can be issues with development of off-flavors, either due to the raw material in question or due to the hydrolysis process itself. This study examined the development of volatile compounds during hydrolysis of hemoglobin. Briefly, porcine hemoglobin was hydrolyzed by 0.5% papain for up to 5 h, and the development of volatile compounds was analyzed via gas chromatography-mass spectrometry. The results showed that there was significant development of a number of volatile compounds with time, e.g., certain Maillard reaction and lipid oxidation products, which are likely candidates for the aroma development during hydrolysis. Furthermore, it was shown that development of a number of the volatiles was due to the hydrolysis process, as these compounds were not found in a control without enzyme.

Monoclonal antibody-based ELISA for the quantification of porcine hemoglobin in meat products

Misusage of porcine blood proteins, such as misbranding and substitution, can cause religious objections, law violation, and food quality concerns. These issues highlight the need for detecting unlabeled or overuse of porcine blood in foods. Compared with acidic and neutral pHs, porcine hemoglobin (PHb) at alkaline pH retained the best solubility, molecular integrity, and immunoreactivity after heat treatment. PHb at acidic and alkaline pHs remained stable during storage at 4 °C for 29 days. A monoclonal antibody (mAb) specific to mammalian hemoglobin, 13F7, was developed. A mAb13F7-based indirect competitive ELISA (icELISA) was optimized for the quantification of PHb in meat products. This assay had a wide working range from 0.5 ppm to 1000 ppm. It was sensitive (limit of detection: 0.5 ppm), precise and reproducible with low inter- and intra-coefficient of variances (<20%). This assay is suitable for government, food industry, and third-party authority to surveillance food quality.

Structure and Function of Adsorbed Hemoglobin on Silica Nanoparticles: Relationship between the Adsorption Process and the Oxygen Binding Properties.

The connection between the mechanisms of protein adsorption on nanoparticles and the structural and functional properties of the adsorbed protein often remains unclear. We investigate porcine hemoglobin adsorption on silica nanoparticles, and we analyze the structural and functional modifications of adsorbed hemoglobin by UV-vis spectrophotometry, circular dichroism, and oxygen binding measurement. The structural analysis of adsorbed hemoglobin on silica nanoparticles reveals a significant loss of secondary structure and a preservation of the heme electronic structure. However, adsorbed hemoglobin retains its quaternary structure and exhibits an enhanced oxygen affinity with cooperative binding. Moreover, the structural and functional modifications are fully reversible after complete desorption from silica nanoparticles at pH 8.7. The tunable adsorption and desorption of hemoglobin on SNPs with pH change, and the full control of hemoglobin activity by pH, temperature, and the addition of inorganic phosphate effectors opens the way to an interesting system whereby protein adsorption on nanoparticles can allow for full control over hemoglobin oxygen binding activity. Our results suggest that adsorption of hemoglobin on silica nanoparticles leads to a new structural, functional, and dynamic state with full reversibility in a way that significantly differs from protein denaturation.

Use of response surface methodology to describe the effect of time and temperature on the production of decoloured, antioxidant and functional peptides from porcine haemoglobin by sub-critical water hydrolysis

Sub-critical water hydrolysis coupled to oxygen injection was used to hydrolyse porcine haemoglobin. The effect through process time of three different temperatures were tested (120–180 °C), under medium pressures (40 atm.). Peptides obtained at 180 °C showed good antioxidant and functional properties, and the best yield (83%). Moreover, the final product presents almost complete discolouration, thus increasing the range of haemoglobin hydrolysates uses in food industry. Regression modelling was used to investigate the main effects of hydrolysis time and temperature. Both were found to be significant. Predicted models were found to be significant (p < 0.05) with high coefficients of determination (R2). This study found that the average peptide size can be predicted from the temperature and process time; besides, evidence seems to show that preferential targets exist to break down the haemoglobin. This method can transform large amounts of proteins into appreciable low-molecular-weight peptides in a simple and fast way.

Effects of resuscitation with polymerized porcine hemoglobin (pPolyHb) on hemodynamic stability and oxygen delivery in a rat model of hemorrhagic shock

The objective of this study was to evaluate the effects of polymerized porcine hemoglobin (pPolyHb) on hemodynamic stability and oxygen delivery in a rat model of hemorrhagic shock. Rats underwent controlled hemorrhage, resulting in the loss of 65% of their blood volume in 90 minutes. The results showed that pPolyHb was superior to hetastarch and saline, and similar to whole blood, in restoring hemodynamic stability and reversing anaerobic metabolism caused by hemorrhage. Furthermore, pPolyHb improved oxygen supply and increased blood oxygen content. These data suggest that pPolyHb can be effective in improving tissue perfusion under conditions of severe hemorrhagic shock.