Sheep Hemoglobin Research Articles

Evaluating sheep hemoglobins with MD simulations as an animal model for sickle cell disease

Sickle cell disease (SCD) affects millions worldwide, yet there are few therapeutic options. To develop effective treatments, preclinical models that recapitulate human physiology and SCD pathophysiology are needed. SCD arises from a single Glu-to-Val substitution at position 6 in the β subunit of hemoglobin (Hb), promoting Hb polymerization and subsequent disease. Sheep share important physiological and developmental characteristics with humans, including the same developmental pattern of fetal to adult Hb switching. Herein, we investigated whether introducing the SCD mutation into the sheep β-globin locus would recapitulate SCD’s complex pathophysiology by generating high quality SWISS-MODEL sheep Hb structures and performing MD simulations of normal/sickle human (huHbA/huHbS) and sheep (shHbB/shHbS) Hb, establishing how accurately shHbS mimics huHbS behavior. shHbS, like huHbS, remained stable with low RMSD, while huHbA and shHbB had higher and fluctuating RMSD. shHbB and shHbS also behaved identically to huHbA and huHbS with respect to β2-Glu6 and β1-Asp73 (β1-Asn72 in sheep) solvent interactions. These data demonstrate that introducing the single SCD-causing Glu-to-Val substitution into sheep β-globin causes alterations consistent with the Hb polymerization that drives RBC sickling, supporting the development of a SCD sheep model to pave the way for alternative cures for this debilitating, globally impactful disease.

Visible-near infrared spectroscopy for detection of blood in sheep faeces

Heavy infestations of the blood-sucking gastrointestinal nematodes, Haemonchus contortus can cause severe anaemia in sheep and leakage of blood into the faeces, leading to morbidity and mortality. Early and accurate diagnosis of infections is critical for timely treatment of sheep, minimizing production and sheep welfare impacts. In pursuit of a quick and easy measure of H. contortus infections, we investigated the use of portable visible near infrared spectrometers for detecting the presence of haemoglobin in sheep faeces as an indicator of H. contortus infection. Calibration models built within the 400–600 nm region by partial least square regression resulted in acceptable prediction accuracies (r2p > 0.70 and root mean squared error of prediction <2.64 µg Hb mg−1faeces) for haemoglobin quantification using two spectrometers. The prediction results from support vector machine regression further improved the prediction of haemoglobin in moist sheep faeces (r2p > 0.87 and root mean squared error of prediction <2.00 µg haemoglobin mg−1faeces). Based on a threshold for anthelmintic treatment of 3 µg Hb mg−1faeces, both the partial least square and support vector machine models showed high sensitivity (89%) and high specificity (>77%). The specificity of the prediction model for detecting haemoglobin in sheep faeces may be improved by adding more variations in faecal composition into the calibration model. Our success in detecting haemoglobin in sheep faeces, following minimal sample preparation, suggests that with further development, vis–near infrared spectroscopy can provide a sensitive and convenient method for on-farm diagnosis of H. contortus infections.

The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation.

The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.

Polymorphism of Pramenka sheep hemoglobin in Central Bosnia

The study of the hemoglobin polymorphism was performed on blood samples, taken from areas inhabited by Pramenka sheep in Central Bosnia: the area of the mountain Vlasic, the upper flow of the river Vrbas and Kupres plateau. Dupska pramenka sheep inhabits the mountain Vlasic and the areas of surrounding municipalities. Privorska pramenka sheep inhabits the area around the upper flow of the river Vrbas and the mountain Vranica and Kupreska pramenka sheep inhabits Kupres plateau. HbA gene frequency was 0.31 and HbB gene frequency was 0.69 for Dupska pramenka sheep. Privorska pramenka sheep had a frequency of 0.33 for the HbA gene, and 0.67 for the HbB gene, in its population. Kupreska pramenka sheep had the lowest frequency of the HbA gene, (0.30), and therefore the maximum frequency of the HbB gene. Hemoglobin genotype frequencies for all investigated types of Pramenka sheep breed are in equilibrium. Investigated types of Pramenka sheep breed inhabit areas adjacent to each other. They are phenotypically quite similar and have a similar frequency of genotype polymorphism of hemoglobin. The difference between them is not statistically significant.

Kinetics of reaction of 5,5′-dithiobis(2-nitrobenzenzoate) with CysF9[93]β sulphydryl group of sheep haemoglobins at neutral and physiological pH ranges

The kinetics of the reaction of 5,5′-dithiobis(2-nitrobenzoate) (DTNB) with CysF9[93]β sulphydryl group of the sheep haemoglobins were studied at neutral and physiological pH (6.8⩽pH⩽7.6) ranges under pseudo first order conditions. Each reaction is of first order with respect to the DTNB concentration. The pH dependence of the observed rate constant of the reaction at neutral and physiological ranges gave a complex trend. The observed rate shows that at neutral pH, the presence of inositol hexakisphossphate (IHP) leads to a relative lowering of the pseudo first order rate constant. Overall, the rate varied among the derivatives as aquomet>oxy>carbomonoxy and the major haemoglobin show a higher reactivity than the minor haemoglobin.

Obtaining protein solvent accessible surface area when structural data is unavailable using osmotic pressure

AbstractHere, we provide an algorithm that predicts solvent accessible surface area (SASA) using concentrated solution osmotic pressure data. Sheep hemoglobin monomer and β‐lactoglobulin are used for verification. Additionally, SASA for structurally unknown calf lens α‐crystallin aggregate is predicted. Using osmotic pressure data, the predicted SASA value for sheep hemoglobin, 22,398 ± 1,244 Å2, was in excellent agreement with computational model predictions (24,304 Å2‐26,100 Å2). Similarly, predicted SASA values for bovine β‐lactoglobulin in pH solutions of pH 5.1, 6.0, and 8.0, were 5,765 ± 1,031 Å2, 6,656 ± 1,082 Å2, and 9,141 ± 1,060 Å2, respectively, were in good agreement with the computationally determined SASA value (7,500 Å2–8,628 Å2). Predicted SASA for the aggregate of calf lens α‐crystallin (800 kDa) was found to be 417,691 ± 16,790 Å2. These results illustrate that this novel method can provide an important experimental alternative in estimating SASA for proteins and, possibly, their complexes in solution. © 2011 American Institute of Chemical Engineers AIChE J, 2012

Tertiary conformational transition in sheep hemoglobins induced by reaction with 5,5´-dithiobis(2-nitrobenzoate) and by binding of inositol hexakisphosphate

We have determined the second-order reverse rate constant, k(R), for the reaction of 5,5 -dithiobis(2-nitrobenzoate) - DTNB - with sheep hemoglobins as a function of pH from values of the second-order forward rate constant, k(F), and the equilibrium constant, K(equ), at 25 degrees C: k(R)=k(F)K(equ). We demonstrate that (i) inositol hexakisphosphate (inositol-P(6)) decreases k(F) and k(R) by increasing K(rt), the rright harpoon over left harpoont tertiary conformation transition constant; (ii) the conformation favored for both the forward and reverse reactions is the r conformation. For stripped hemoglobin we obtain from the k(F) data a t isomer population of 34.6% (+/-14) prior to reaction with DTNB; from the k(R) data we calculate a t isomer population of 44.8% (+/-4) following reaction with DTNB. In the presence of inositol-P(6) the latter value is increased to 79.5% (+/-2). These results demonstrate that an allosteric transition occurs on reaction with DTNB and on inositol-P(6) binding.

Transition of haemoglobin between two tertiary conformations: Inositol hexakisphosphate increases the transition constant and the affinity of sheep haemoglobin for 5,5′-dithiobis(2-nitrobenzoate)

The equilibrium constant ( K equ) for the reaction of 5,5′-dithiobis(2-nitrobenzoate) — DTNB — with the CysF9[93]β sulphydryl group of the haemoglobins of the sheep decreases by about two orders of magnitude between pH ≈ 5.6 and 9.2: from a mean of 7.2 ± 1 to a mean of 0.044 ± 0.01. Calculations from the pH dependence of K equ show that in the r ⇌ t tertiary conformational transition of haemoglobin the t isomer population is 50.7 and 61.8% for the major and minor haemoglobins, respectively. In the presence of inositol hexakisphosphate (inositol-P 6), K equ increases for both haemoglobins by about an order of magnitude through most of the pH range. The t isomer population also increases to 82.1 and 79.6% for the major and minor haemoglobins, respectively. These results indicate that inositol-P 6 increases the affinity of the sulphydryl for DTNB by increasing the population of the t isomer. It is highly probable that a minimum four-state model that includes the r ⇌ t transition is required for a full understanding of haemoglobin function.

Iron Acquisition by Ornithobacterium rhinotracheale

Ornithobacterium rhinotracheale (ORT) is an emerging respiratory pathogen of poultry in North America that is causing millions of dollars in economic losses to the poultry industry. Ornithobacterium rhinotracheale is associated with airsacculitis, pleuritis, pneumonia, and consolidation of lungs. Little is known about the molecular mechanisms of infection. In this study, the mechanism of iron acquisition by O. rhinotracheale was explored. O. rhinotracheale strains grown under iron deprivation in media containing 200 microM 2,2'-dipyridyl did not secrete siderophores as measured by the chrome azurol S (CAS) agar and CAS solution assays. Filter disks impregnated with various protein-bound iron compounds and inorganic iron salts of Fe(III) and Fe(II) placed on iron-restricted agar inoculated with a lawn of O. rhinotracheale supported growth from sheep and porcine hemoglobins, ovotransferrin, Fe(III), and Fe(II), but they did not support growth from bovine transferrin, bovine apo-transferrin, bovine lactoferrin, and hemin. However, both bovine hemoglobin and transferrin supported growth of O. rhinotracheale serotype C. Four immunoreactive proteins involved in iron acquisition were identified in an O. rhinotracheale membrane extract by using mass spectrometry. Furthermore, O. rhinotracheale field strains showed differential sensitivity to 2,2'-dipyridyl. Of the 72 field strains tested, 22 strains were resistant to the iron chelator at concentrations of 50 microM and 100 microM, suggesting this attribute may be related to disease-producing potential of these strains. This is the first report on the identification of the iron acquisition mechanism of O. rhinotracheale.

Structure and function of sheep hemoglobin Chios: A novel allele at the HBBB locus with two Lys → Arg substitutions at positions β66(E10) and β144(HC1)

A novel hemoglobin variant was observed in pure sheep ( Ovis aries) breeds of the island of Chios (Greece), Egypt and Hungary. This silent variant was identified by gel electrophoresis and RP-HPLC of dissociated globin chains. Two Arg for Lys substitutions were detected, by means of MALDI TOF electrospray mass spectrometric analysis for the intact globins, at positions β66(E10) and β144(HC1) of a globin chain having the sequence of the β B chain. Sequencing of the β-globin gene confirmed the variant gene as being an allele of the HBBB locus having the AAG → AGG and the AAA → AGA mutations at codons 66 and 144, respectively, both corresponding to the Lys → Arg substitution. The intrinsic oxygen affinity of the variant Hb (log P 50 = 0.79 at pH 7.0) was found to be intermediate between that of the sheep Hb B (log P 50 = 0.92) and that of Cypriot mouflon ( O. a. ophion) Hb (log P 50 = 0.53), the latter having only the Lys → Arg change at β144, whereas nearly no differences were observed in the presence of the Cl − physiological effector. Result supports the indication that Arg at β144 enhances the role of the ligand in decreasing oxygen affinity, this effect being partially counteracted when Arg is at β66. Data also shows that the Lys → Arg change at β66 is responsible for 1.49 fold reduction in the intrinsic oxygen affinity. This hitherto undescribed variant increases to seven the number of alleles at the sheep HBBB locus. Following the nomenclature used for human Hb variants, the new allele was termed as the Hb Chios or [β B66(E10) Lys → Arg, 144(HC1)Lys → Arg], whereas the proposed genetic nomenclature of the locus is HBBK.

Studies on Haemoglobin Polymorphism of Two Breeds of Iranian Sheep and Its Relationship to Concentrations of Iron, Copper, Haemoglobin, Haematocrit and RBC number

After electrophoresis on cellulose acetate, two haemoglobin phenotypes were detected in Baloochi and Kordi breeds: AA and AB phenotypes. AA was commonest in two breeds. The incidence of type AB haemoglobin in Baloochi and Kordi breeds was 26.5% (9/34) and 9.5% (2/21), respectively. BB phenotype was not seen in Baloochi and Kordi breeds. In sheep with AB phenotype, haemoglobin B was dominant. The mean +/- SD of the two kinds of haemoglobin in sheep with AB phenotype were haemoglobin B percentage 60.5% +/- 9.04%, haemoglobin B absolute 73.84 +/- 5.44 g/L, haemoglobin A percentage 39.5% +/- 9.04%, haemoglobin A absolute 32.88 +/- 2.89 g/L. There were no significant differences for total haemoglobin, haematocrit, red blood cell (RBC) number, iron and copper levels between breed, sex and age groups and also between sheep with AA phenotype and AB phenotype. Pearson's method showed significant correlations for total haemoglobin with packed cell volume (PCV), RBC number, copper concentration and RBC number with PCV, copper level and PCV with copper amount and copper concentration with iron level (p<0.05). In the Kordi breed, significant correlations were seen for total haemoglobin with PCV, RBC number, copper concentration and PCV with RBC number and RBC number with copper level and copper concentration with iron amount (p<0.05). In the Baloochi breed, significant correlations were detected for total haemoglobin with PCV, RBC number and PCV with RBC number (p<0.05).

The Phenotype of Sheep Hemoglobins Containing Distinct α Chains Is Influenced by β Chain Genotype

In sheep carrying simultaneously extra α‐globin genes (triplications and quadruplications) and two α‐chain allelic variants, a gradient of decreasing expression of the downstream genes was previously reported. We show here that in these sheep the proportions of the normal and variant Hbs reflect not only the position effect, but also the differential affinity of one of the α variants for the βA and βB allelic chains.

Anthelmintic activity of crude aqueous extract of Nauclea latifolia stem bark against ovine nematodes

The anthelmintic efficacy of Nauclea latifolia stem bark aqueous extract was studied in sheep with natural acute/sub-acute parasitic gastro-enteritis due primarily to mixed nematode species. Graded doses of the extract (400, 800 and 1600 mg/kg, p.o for 5 consecutive days) significantly reduced faecal egg counts in infected animals. The percentage reduction (93.8%) by 1600 mg/kg of the extract was comparable to that of 5 mg/kg of albendazole (94.1%). The administration of the extract resulted in improved haemoglobin and leucocytosis values in worm-infected sheep.

Comparison of the haemodynamic effects of nitric oxide synthase inhibition and nitric oxide scavenging in endotoxaemic sheep

The present study compared the effects of nitric oxide (NO) synthase inhibition and NO scavenging with haemoglobin in endotoxaemic sheep. 12 sheep were instrumented for chronic study. Six sheep received LG-nitro-arginine-methylester (L-NAME, 2.5 mg/kg bolus followed by a continuous infusion of 0.5 mg/kg per h), the other 6 sheep received pyridoxalated haemoglobin polyoxyethylene conjugate (PHP, 100 mg/kg bolus followed by a continuous infusion of 20 mg/kg per h). Haemodynamic and oxygenation parameters were measured in healthy sheep, after infusion of Salmonella typhosa endotoxin (10 ng/kg per min) for 24 h and after infusion of L-NAME or PHP. The infusion of endotoxin resulted in a hypotensive, hyperdynamic circulation. Infusion of L-NAME increased mean arterial pressure (MAP) from 76.1 +/- 4.2 mmHg to normal values of 95.8 +/- 5.7 mmHg (p < 0.05). PHP increased MAP from 73.0 +/- 3.0 to 88.6 +/- 4.7 mmHg (p < 0.05). This increase in MAP was associated in the L-NAME group with a more prominent drop in cardiac index (from 10.2 +/- 0.4 to 7.0 +/- 0.51.min-1.m-2; p < 0.05) than in the PHP group (from 10.7 +/- 0.2 to 9.3 +/- 0.61.min-1.m-2). During the first 90 min of infusion, cardiac index remained lower in the L-NAME group than in the PHP group. The increase in pulmonary vascular resistance was also higher in the L-NAME group. These results suggest, that at the doses used in the experiment, NO scavenging with PHP has smaller effects on cardiac index and pulmonary vascular resistance than NO synthase inhibition with L-NAME. Therefore, the concept of NO scavenging in hyperdynamic sepsis should be further evaluated.

A Comparative Study on the Functional Properties of the Wild European Mouflon and Domestic Sheep Hemoglobins

The functional properties of Hb B of the wild European mouflon ( Ovis gmelini musimon), Hb B of domestic sheep ( Ovis aries), and Hb C isolated from anemic mouflon were investigated. Mouflon and sheep Hbs appear to be very similar in their response to organic anions and protons, whereas sheep Hb B displays an oxygen affinity lower than that of mouflon Hb B and sheep Hb A. Mouflon Hb B and Hb C, like sheep Hb A and Hb C, have similar efficiencies in transporting oxygen to the tissues. As in other ruminant Hbs, the effect of temperature on the oxygen affinity is slight. Data suggest that mouflon Hb B is not only structurally, but even functionally, more similar to sheep Hb A than to sheep Hb B.

The alpha-hemolysin of Streptococcus gordonii is hydrogen peroxide.

The alpha-hemolysin of viridans group streptococci, which causes greening of intact erythrocytes, is a potential virulence factor as well as an important criterion for the laboratory identification of these bacteria; however, it has never been purified and characterized. The alpha-hemolysin of Streptococcus gordonii CH1 caused characteristic shifts in the A403, A430, A578, and A630 of sheep hemoglobin. A spectrophotometric assay was developed and used to monitor purification of alpha-hemolysin during extraction in organic solvents and separation by reverse-phase high-performance liquid chromatography (HPLC). The alpha-hemolysin was identical to hydrogen peroxide with respect to its effects on erythrocyte hemoglobin, oxygen-dependent synthesis by streptococci, insensitivity to proteases, inactivation by catalase, differential solubility, failure to adsorb to ion-exchange chromatography resins, and retention time on a reverse-phase HPLC column. The amount of hydrogen peroxide present in HPLC-fractionated spent culture medium was sufficient to account for all alpha-hemolytic activity observed.

Antagonism of teicoplanin and vancomycin activity by chocolatized Mueller-Hinton agar.

Chocolatized (80 degrees C, 13 min) Mueller-Hinton agar antagonized the inhibitory activities of teicoplanin and vancomycin against reference strains of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. This antagonism was due to heat-exposed sheep erythrocytes, sheep hemoglobin, and the supernatant fluid from lysed sheep erythrocytes, but not to sheep serum. Neither water-soluble cholesterol, bovine albumin, bovine serum, hematin, hemin nor egg yolk suspension antagonized teicoplanin and vancomycin.

Evaluation of novel vancomycin-containing medium for primary isolation of Kingella kingae from upper respiratory tract specimens.

A new selective medium (BAV), consisting of trypticase agar with 5% sheep hemoglobin and 2 micrograms of vancomycin per ml, was compared with the routine blood-agar medium for the primary isolation of Kingella kingae from upper respiratory specimens from a population of young children. Infection was detected by the BAV medium in 43 of 44 (98%) cultures positive for K. kingae, and detection of the organism was facilitated by inhibition of gram-positive flora. Infection was detected in only 10 of 44 (23%) positive cultures by the blood-agar medium, and plates were usually covered by abundant normal flora, making the recognition of K. kingae much more difficult. Challenge of the medium with different organisms of respiratory origin showed that the BAV medium was inhibitory for gram-positive cocci and Haemophilus influenzae but that it supported growth of eight K. kingae strains isolated from patients with invasive infections. The new medium appears to be a useful epidemiological tool for studying the respiratory carriage of K. kingae.

Stability of haem pigments in model systems and cooked meat

The stability of sheep haemoglobin and myoglobin in aqueous solution at 80, 100 and 121°C for 1 h was measured by subjecting portions of the heated solutions to electrospray mass spectrometry (ESMS). ESMS dissociates haem proteins into the globin chains and the haem moiety and, with haemoglobin, degradation of the α-(15047·5 Da) and β-(16073·3 Da) chains was observed at all heating temperatures. Under the same conditions, sheep myoglobin dissociated into the globin (16923·2 Da) and haem parts but the globin was stable and few degradation products were observed in the ESMS trace (mass range 4–20 kDa) even after 1 h at 121°C. There did seem to be limited breakdown of the globin due to loss of 170 Da. From the amino acid sequence, it is postulated that this is due to loss of GLY-LEU from the N-terminus. Methods for extracting myoglobin from raw and cooked meat were then investigated. Water was adequate for myoglobin extraction from raw meat but urea solution was required for adequate extraction of cooked meat samples. Sheep meat was heated at 80, 100 and 121°C in sealed cans, extracted and the mass profile in the range 4–20 kDa measured. Myoglobin was the major peak when samples were heated for 10, 20, 30 and 40 min. After that time, other peaks appeared although the myoglobin globin chain was still apparent. The results are discussed in relation to using myoglobin as a marker for meat speciation.

Sheep haemoglobin i or β b13(a10)gly → ser: an example of a cpg mutation in vertebrates. Characterization using fab-mass spectrometry and amino acid sequencing

1.1. The amino acid substitution which characterizes the haemoglobin I variant from sheep has been ascertained using a combination of Fast Atom Bombardment mass spectrometry and protein sequencing.2.2. A Ser for Gly substitution at position 13 (10 of the A helix) was found in a polypeptide with the overall sequence of the βB globin.3.3. On the basis of the nucleotide sequence of the βB-globin gene, a C to T transition occurring on a CpG doublet is considered to be responsible for the amino acid substitution.4.4. This represents the first observation of a variant sheep Hb due to a mutation which is rather common in the human genome.5.5. Amongst ruminants, serine is normally present at position 13 of goat and sheep ε11 and γ chains and of bovine γ chain which had an independent and more ancient evolutionary origin than the β chains.