Porcine Hemoglobin Research Articles

Porcine Hemoglobin Hydrolysate as a Biostimulant for Lettuce Plants Subjected to Conditions of Thermal Stress

The effects of different kinds of biotic and abiotic stress on crops can be lessened through exogenous application of different biostimulant products. Although some of these products come from enzymatic hydrolysates derived from animal remains, the literature does not contain references to the use of enzymatic hydrolysates obtained from animal hemoglobin, specifically porcine blood. With the aim of evaluating the effectiveness of a product obtained from the enzymatic hydrolysis of porcine hemoglobin (PHH) as a biostimulant that lessens the effects of thermal stress, two experiments were carried out in which lettuce plants (Lactuca sativa) were subjected to short-term episodes of intense cold and heat. After these episodes, different doses of the PHH product were administered into the growing medium. Moreover, in the heat episode experiment, one group of plants was first subjected to a heat episode and then administered a commercial biostimulant with the aim of comparing its efficacy with the PHH product. The biometric measurements carried out on the lettuce plants several days after being subjected to the episodes of cold and heat and then either administered or not administered the indicated treatments show that at the highest tested dose, the PHH product promoted a reaction that lessened the harmful effects caused by the intense cold and heat treatments.

Hämoglobin-Hyperpolymere, künstliche Sauerstoffträger eines neuen Typs - Konzept und aktueller Stand der Entwicklung

In the clinical setting, artificial oxygen carriers are needed when a patient has a tissue oxygen deficiency which he/she can not automatically compensate. There are two quite different situations where this might occur: (1) Heavy blood loss (e. g., following an accident) and (2) insufficient perfusion (e. g., as a result of arteriosclerosis or myocardial infarction) or anaemia, both without blood loss. In the first instance, an iso-oncotic oxygen-transporting plasma expander is required, whereas in the second instance a (hypo-oncotic) so-called blood additive is needed. This second type of situation also presents the greater range of very important indications. Experimental work has shown that, in comparison to erythrocytes, dissolved haemoglobin is able to release oxygen more rapidly (effective plasmatic transport), while at the same time also facilitating oxygen release from erythrocytes (mediator function). Blood additives occur naturally in lower forms of life (e. g., earthworm) where they can be found in the form of giant oxygen-carrying molecules. Using these natural forms as a basis, new oxygen-transporting blood additives were designed and developed (so-called haemoglobin hyperpolymers: HP (3)Hb) which exhibit a strong oxygen affinity (half saturation partial pressure p (50) = 16 Torr) and high cooperativity (n (50) = 2.1). One product has, up until now, been produced aseptically on a small technical scale and consists of highly purified, polymerised and pegylated porcine haemoglobin which is free of monomers and oligomers, with a mean molecular weight of approximately 800 kDa. It is sufficiently low in endotoxin (< 0,029 EU/mL), blood plasma compatible, and - at an effective concentration of 3 g/dL in blood plasma - causes only minor increases in oncotic pressure or viscosity. The product has a shelf-life of up to 2 years and is administered as a carbonyl derivative. Its half-life in the conscious rat is 30 h. This product was found to prevent death in rats where acute lung injury was induced using oleic-acid. In human self-experiments this product was repeatedly administered: No effects on blood pressure and heart rate, no increase in blood transaminase concentration and no immunological reaction were seen; the latter was also not found in selected sensitive mice. Furthermore, the blood additive is universally applicable as an oxygen transporter, since, when mixed with a conventional plasma expander, it can also be used to treat an oxygen deficiency occurring together with blood loss.

Chemically modified porcine hemoglobins and their biological properties.

Hemoglobin cross-linked with small molecular modifiers turns out to be more stable. Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge the molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions. In the present study, the optimal conditions for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl) fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb (DBBF-pHb) showed improved oxygen affinity and the ability to resist the dissociation of the alpha2beta2 tetramer compared with the natural protein. DBBF-pHb was then bound to the activated PEG. The results indicated that the pHb modified with DBBF and PEG had more stable tetrameric conformation with a molecular weight of 107000. Their oxygen half-saturation pressure (P50) is around 3.33 kPa, which approximates the physiological P50 of human red blood cells. Both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by enzyme-linked immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the treatment of hemorrhagic shock where modified hemoglobin solutions were used as resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model.

Two TonB systems in Actinobacillus pleuropneumoniae: their roles in iron acquisition and virulence.

Iron acquisition in vivo by Actinobacillus pleuropneumoniae depends upon a functional TonB system. Tonpitak et al. (W. Tonpitak, S. Thiede, W. Oswald, N. Baltes, and G.-F. Gerlach, Infect. Immun. 68:1164-1170, 2000) have described one such system, associated with tbpBA encoding the transferrin receptor, and here we report a second, termed tonB2. This gene cluster (exbB2-exbD2-tonB2) is highly homologous to those in other Pasteurellaceae, unlike the earlier system described (now termed tonB1), suggesting that it is the indigenous system for this organism. Both tonB2 and tonB1 are upregulated upon iron restriction. TonB2, but not TonB1, was found to be essential for growth in vitro when the sole source of iron was hemin, porcine hemoglobin, or ferrichrome. In the case of iron provided as iron-loaded porcine transferrin, neither tonB mutant was viable. The tonB1 phenotype could be explained by a polar effect of the mutation on transcription of downstream tbp genes. We propose that TonB2 is crucial for the acquisition of iron provided in this form, interacting with accessory proteins of the TonB1 system that have been demonstrated to be necessary by Tonpitak et al. TonB2 appears to play a much more important role in A. pleuropneumoniae virulence than TonB1. In an acute porcine infection model, the tonB2 mutant was found to be highly attenuated, while the tonB1 mutant was not. We hypothesize that acquisition of the tonB1-tbp gene cluster confers a biological advantage through its capacity to utilize transferrin-iron but that TonB1 itself plays little or no part in this process.

New artificial oxygen carriers made of pegulated polymerised pyridoxylated porcine haemoglobin (P 4Hb)

Oxygen-carrying plasma expanders are designed for use as iso-oncotic ‘blood substitutes’ to combat oxygen deficiencies caused by blood loss. In contrast, a hypo-oncotic artificial oxygen carrier can be added to existing blood — as a ‘blood additive’. It has potential therapeutic use for deficiencies of oxygen which are not entailed by blood (volume) lack, and can therefore not be treated by a ‘blood substitute’, e.g. anaemias, local ischaemias and their complications such as stroke or myocardial infarction, or lack of oxygen in tumours, reducing the effectiveness of anti-cancer treatments by irradiation or chemotherapy. For such a novel approach haemoglobin-based oxygen-carrying additive, the haemoglobin must be highly polymerised in order to decrease the oncotic pressure, which can be received many times lower compared with smaller molecular size haemoglobins. Our aim is to produce haemoglobin polymers with narrow distributions of molecular weights of approximately 1 000 000 g/mol, preferably produced in high yield and at low cost. But polymerising haemoglobin by cross-linking normally results in a so-called percolation distribution of molecular weights, with a large amount of insoluble material, and with only poor yields of the desired polymers. A newly developed one-vessel synthesis procedure, which includes a controlled marked dilution of the synthesis medium during the cross-linking reaction, enables yields of polymerised haemoglobin (P 4Hb) of over 80 %. Those preparations are easy and cheap to perform at large scales. P 4Hb hyperpolymers (the high molecular moiety of P 4Hb) are suitable for an oxygen-carrying blood additive: their oxygen-binding properties are sufficient, they are fully compatible with human blood plasma, and at the intended therapeutic concentration of approximately 30 g/l oncotic pressures are very low, and the impact on blood viscosity is tolerable.

A model for evaluation of artificial oxygen carriers regarding circulation, respiration and metabolism in anesthetized spontaneously breathing guinea pigs.

The evaluation of artificial oxygen carriers requires experiments with suitable animals. Many investigators do this with the classical laboratory animal, the rat, but it has a quite different oxygen pressure of half saturation (p50 = 36 mmHg) from that of humans (26 mmHg). It was demonstrated that induced changes of the p50 value in animals provokes substantial changes in important cardiovascular parameters. Therefore, we decided to develop a guinea pig model for evaluation of artificial oxygen carriers, because it has a p50 of about 24 mmHg that is very similar to that of man. We found an anesthesia combination using fentanyl/droperidol/urethane to be very suitable for the narcosis, because important cardiovascular and respiratory parameters remain normal. Our model allows assessment of arterial pressure, oxygen uptake, carbon dioxide release, cardiac output, total peripheral resistance, shock index, blood lactate level, and, in particular, it allows to differentiate the oxygen transport in blood. We evaluated two hemoglobin-based oxygen carriers: bovine and porcine hemoglobin polymerized with glutaraldehyde in isoncotic solution (n = 3). Control experiments (n = 4) were done with an isotonic albumin solution. The protocol comprised a so-called exchange phase (I) with different degrees of hemodilution and a so-called time phase (II), an observation period with a hematocrit of 10% over about 3 hours. In the control group substantial changes in mean arterial pressure, total peripheral resistance, shock index, oxygen uptake and of blood lactate level were seen. All these effects were prevented by the artificial oxygen carriers tested. The carriers proved to be very effective, as small quantities in blood effectively restored these parameters, presumably via synergetic effects. Moreover, the experiments clearly revealed the limitation of hemodilution, at least in guinea pigs: below a hematocrit of 20% all parameters mentioned above changed significantly. The animal model presented proved to be appropriate for the evaluation of artificial oxygen carriers.

Absorbance Spectra of Inter‐Species Hemoglobins in the Visible and Near Infrared Regions

SummaryThis study was initiated to determine if the absorbance spectra of hemoglobins from nonhuman species were sufficiently different from those of human hemoglobin to affect the applicability of pulse oximetry to common domestic species. Hemoglobin was isolated from the blood of humans, dogs, cats, horses, cows, and pigs. The millimolar extinction coefficients for oxygenated and deoxygenated hemoglobin samples were determined over the wavelength range of 600 to 1000 nm. Minor differences in the absorbance of both oxyhemoglobin and deoxyhemoglobin were found between human hemoglobin and that of the dog, cat, horse, cow, and pig. However, correction for the presence of methemoglobin, the effects of light scattering by impurities in the solution, and technical error rendered these differences statistically insignificant. The results of this study suggest that the spectral properties of canine, feline, equine, bovine, and porcine hemoglobin do not differ significantly from that of human hemoglobin and should not be a limiting factor in the application of pulse oximeters that are based on human algorithms to animal species.

Molar masses and structure in solution of haemoglobin hyperpolymers--a common calibration of size exclusion chromatography of these artificial oxygen carriers.

We are developing artificial oxygen carriers for medical use, based on synthetic polymers--so-called hyperpolymers--obtained by cross-linking mammalian haemoglobins. One requirement with respect to the polymers is that they should not increase the oncotic pressure of blood remarkably--this can be realized by high molecular weights of the polymers with a narrow distribution. They may act as a oxygen transporting blood additive, and--in combination with a plasma expander--as a blood substitute. Another important and desired property of the artificial oxygen carrier is a low viscosity, which--first--is due to a high degree of uniformity of the polymer size (or molar mass) distribution and--second--is influenced by the so-called structure in solution of the haemoglobin hyperpolymers. In this paper former determinations of molar masses--with size exclusion chromatography (SEC)--and of the structure in solution--using viscometric measurements--of hyperpolymers of human haemoglobin, synthetized with glutaraldehyde and with bis(thioisocyanato) benzenesulfonic acid as cross-linkers, were extended to hyperpolymers of bovine and pyridoxylated porcine haemoglobin, cross-linked with glycolaldehyde. These determinations were done by applying a new iterative procedure. Within a range of error all SEC calibration curves found were the same for all hyperpolymers investigated. So-called MARK-HOUWINK or structure in solution diagrams (logarithm of intrinsic viscosity versus logarithm of molar mass) yield equal straight lines for all the haemoglobin polymers. The first derivatives of these lines are the MARK-HOUWINK exponents which has a mean value of 0.38. These results indicate that there exists a common SEC calibration line for all different polymer haemoglobins produced with comparable preparative procedures. This calibration line differ significantly from that of native globular proteins--haemoglobin hyperpolymers are less compact--so a calibration of SEC with globular proteins for the determination of molar masses of haemoglobin polymers is erroneous. Furthermore, the structure in solution of the hyperpolymers is clearly different from that of flexible, randomly coiled, linear artificial polymers: hyperpolymers are more compact. A possible explanation is that the hyperpolymers--according to a great number of functional amino groups of haemoglobin-contain many intra-polymeric cross-links, and thus are at least import branched polymers or even macromolecular networks of the constituting haemoglobin "monomers".

Structure Determination of Aquomet Porcine Hemoglobin at 2.8 Å Resolution

Considerable attention is currently focused on hemoglobins from lower mammals, such as the pig, for potential use in cell-free blood substitute preparations safe for use in humans. As the first step in the elucidation of structure-function relationships in porcine hemoglobin, we have determined the three-dimensional structure of aquomet porcine hemoglobin at 2.8 Å resolution. Overall, the porcine hemoglobin tetramer is structurally similar to that of human oxyhemoglobin, and the r.m.s. deviation of all backbone atoms (minus five residues at the amino and carboxyl termini of each subunit) is 0.8 Å. This similarity is not unexpected given that human and porcine hemoglobins exhibit 85% sequence identity. However, regions of subtle structural differences are implicated in subtle functional differences between the two proteins, such as the 20 to 25% inhibition of the alkaline Bohr effect and the accompanying reduction in oxygen-linked chloride binding observed for porcine hemoglobin. The structural similarity of these two mammalian hemoglobins also rationalizes the novel hybridization behavior of pig and human subunits in transgenic pigs expressing both porcine and human hemoglobins in porcine erythrocytes.

Production of functional human hemoglobin in transgenic swine.

A construct containing the locus control region (LCR) from the human beta globin locus together with two copies of the human alpha 1 gene and a single copy of the human beta A gene was used to obtain three transgenic pigs. The transgenic pigs are healthy, not anemic, and grow at a rate comparable to non-transgenic littermates. All animals expressed the human genes. However, alpha globin was consistently expressed at higher levels than beta globin. Isolation of the human hemoglobin from both porcine hemoglobin and other non-hemoglobin proteins was accomplished by ion exchange chromatography. The purified porcine derived human hemoglobin exhibited an oxygen affinity similar to that of human derived human hemoglobin.

491—Linear dependence of polarographic catalytic current on the numeber of SH groups in the molecule for haemoglobins of different origin

Polarographic catalytic currents for solutions containing bovine, ovine, dog, human, rabbit, porcine or pigeon haemoglobins, and bovine or human methaemoglobins at several NH 3 and CoCl 2 concentrations were studied. At earlier elaborated conditions [9], for isomolar solutions of five haemoglobins of known amino-acid composition, a linear correlation between the current of the first catalytic wave and the number of SH groups in the molecule was obtained, this being confirmed by a very high value of the correlation coefficient. No correlation could be stated using solutions of lower ammonia concentrations for the first and, under all conditions studied, for the second catalytic wave. On the basis of the linear dependence obtained, a number of SH groups in tetramers—and hence in the ß-chain of dog haemoglobin and the α-chain of porcine haemoglobin—were found. The fact that the straight lines obtained do not intersect with the origin of coordinates is briefly discussed. Values of the standard deviation pointed to the use of low cobaltous chloride concentrations in the solution. It has also been stated that the magnitude of the catalytic current of haemoglobins depends on their methaemoglobin content, which should be connected with structural differences in these two species.

Linear dependence of the polarographic catalytic current on the number of SH groups in the molecule for haemoglobins of different origin

Polarographic catalytic currents for solutions containing bovine, dog, human, rabbit, porcine or pigeon haemoglobins, and bovine or human methaemoglobins at several NH 3 and CoCl 2 concentrations were studied. At earlier elaborated conditions [9], for isomolar solutions of five haemoglobins of known amino-acids compositions, a linear correlation between the current of the first catalytic wave and the number of SH groups in the molecule was obtained, this being confirmed by a very high value of the correlation coefficient. No correlation could be stated using solutions of lower ammonia concentrations for the first and, under all conditions studied, for the second catalytic wave. On the basis of the linear dependence obtained, a number of SH groups in tetramers—and hence in the β-chain of dog haemoglobin and the α-chain of porcine haemoglobin—were found. The fact that the straight lines obtained do not intersect with the origin of coordinates is briefly discussed. Values of the standard deviation pointed to the use of low cobaltous chloride concentrations in the solution. It has also been stated that the magnitude of the catalytic current of haemoglobins depends on their methaemoglobin content, which should be connected with structural differences in these two species.

Isolation and structure of several peptides from porcine hypothalami

Ten peptides were isolated from porcine hypothalami and structurally elucidated. These included four dipeptides Arg-Phe, Phe-Tyr, Val-Trp, and Tyr-Phe; a tripeptide Lys-Phe-Tyr; two tetrapeptides Gly-Lys-Val-Asn and Phe-Glu-His-Glu, a nonapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; a decapeptide Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe and a hexadecapeptide Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe-Asn-Leu. The tetrapeptide Gly-Lys-Val-Asn, the nonapeptide, the decapeptide and the hexadecapeptide most probably represent artifact fragments of α- and β-chains of porcine hemoglobin. The natural or synthetic Gly-Lys-Val-Asn and Phe-Glu-His-Glu had some growth hormone releasing activity while Val-Trp, Tyr-Phe and Lys-Phe-Tyr had slight prolactin releasing activity. The biological activities of other peptides have not been determined yet.

Isolation, structural elucidation and synthesis of a tetradecapeptide with in vitro ACTH-releasing activity corresponding to residues 33–46 of the α-chain of porcine hemoglobin

A peptide found in acetic acid extracts of porcine hypothalami and capable of stimulating the release of ACTH in vitro was isolated in pure state, structurally identified as Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Pre-Pro-His-Phe and synthesized. This tetradecapeptide, which corresponds to amino acid residues no. 33–46 in the sequence of the α-chain of porcine hemoglobin, probably represents an artefact of extraction or isolation procedures. Since this peptide stimulates ACTH release from rat pituitary fragments and from monolayer cultures of pituitary cells, but not in vivo , caution must be exercised in interpreting the results of in vitro assays for corticotropin releasing factor.

Synthesis of a proposed growth hormone releasing factor

The synthesis of Val-His-Leu-Ser-Ala-Glu-Glu-Lys-Glu-Ala, isolated by Schally, et al. from porcine hypothalamus and reported by him to possess growth hormone releasing activity, is described. Similarities between this decapeptide and the amino-terminal sequence of the β-chain of porcine hemoglobin are pointed out. The syntheses of two analogs including the amino-terminal decapeptide of the β-chain of human hemoglobin are also described.