Two-nucleotide codon change in a hemoglobin polymorphism of the Celebes black ape (Macaca nigra)

A hemoglobin polymorphism involving variant -chains was demonstrated in the Celebes black ape, Macaca nigra. Fingerprinting and amino acid analysis of the tryptic peptides from the two chain types have shown that they differ by a single amino acid substitution, between lysine and aspartic acid, which requires a two-nucleotide change in the corresponding codon. Another substitution in the same codon is found as a species difference between the black ape -chain and that of other macaques.     

Glycosylated equine prolactin and its carbohydrate moiety

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities,G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about l/30th the mitogenic activity of bovine PRL; G-ePRL was approximately l/10th as active as ePRL. Glycosylation of G-ePRL at Asn³¹ was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29–37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man₃, GlcNAc₂, GalNAc₁, Fuc_0.6, Gal_0.2, NeuAc_0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn³¹ carbohydrate unit is a fucosylated complex mono- and/or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.     

Esterase activity and interaction of human hemoglobin with diclofenac sodium: A spectroscopic and molecular docking study

To get an idea about the pharmacokinetics and pharmacodynamics, it is important to study the drug‐protein interaction. Therefore, herein, we studied the interaction of diclofenac sodium (DIC) with human hemoglobin. The binding study of nonsteroidal antiinflammatory drug, DIC with human hemoglobin (HHB) was done by utilizing fluorescence, UV–visible, time‐resolved fluorescence and far‐UV circular dichroism spectroscopy (CD). Various thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) were also calculated. CD results showed that DIC induces secondary structure change in HHB. Fluorescence resonance energy transfer was also performed. Additionally, it was also observed that DIC inhibits the esterase‐like enzymatic activity of HHB via competitive inhibition.     

表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*

透明颤菌血红蛋白基因vgb在多种研究和工业发酵菌中异源表达很好的解决了高密度发酵中的溶氧率问题。酿酒酵母是经典的真核模型,且在发酵工业中具有重要的应用价值,但vgb在酿酒酵母中异源表达对细胞生长的影响并不清楚。以ADH1为启动子构建了含透明颤菌(Vistreoscilla)血红蛋白基因vgb的异源表达质粒YEplac195-ADH1pr-vgb,并转化至酿酒酵母BY4741。通过生长敏感性实验,发现在发酵碳源和非发酵碳源中,vgb的异源表达均抑制了菌株生长。接着,通过2',7'-二氯荧光黄双乙酸盐和PI染色和脂质过氧化产物检测分析,发现过表达vgb的酿酒酵母细胞中活性氧(ROS)的积累、细胞膜通透性改变以及脂质过氧化。结果表明,酿酒酵母中过表达vgb改变细胞的氧化状态促进活性氧的累积,氧化应激导致菌株的生长抑制。     

Formation of Free Radicals and Nitric Oxide Derivative of Hemoglobin in Rats During Shock Syndrome

Free radicals have been postulated to play an important role as mediators in the pathogenesis of shock syndrome and multiple-organ failure. We attempted to directly detect the increased formation of radicals by Electron Spin Resonance (ESR) in animal models of shock, namely the endotoxin (ETX) shock or the hemorrhagic shock of the rat. In freeze-clamped lung tissue, a small but significant increase of a free radical signal was detected after ETX application. In the blood of rats under ETX shock, a significant ESR signal with a triplet hyperfine structure was observed. The latter ESR signal evolved within several hours after the application of ETX and was localized in the red blood cells. This signal was assigned to a nitric oxide (NO) adduct of hemoglobin with the tentative structur ((a2+ NO)/23+)2. The amount of hemoglobin-NO formed, up to 0.8% of total hemoglobin, indicated that under ETX shock a considerable amount of NO was produced in the vascular system. This NO production was strongly inhibited by the arginine analog N^G-monomethyl-arginine (NMMA). The ESR signal of Hb-NO was also observed after severe hemorrhagic shock. There are three questions, namely (i) the type of vascular cells and the regulation of the process forming such a large amount of NO during ETX shock, (ii) the pathophysiological implications of the formed NO, effects which have been described as cytotoxic mediator, endothelium-derived relaxing factor (EDRF) or inhibitor of platelet aggregation, and (iii) the possible use of Hb-NO for monitoring phases of shock syndrome.     

Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin

Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Volumetric properties underlying ligand binding in a monomeric hemoglobin: A high-pressure NMR study

The 2/2 hemoglobin of the cyanobacterium Synechococcus sp. PCC 7002, GlbN, coordinates the heme iron with two histidines and exists either with a b heme or with a covalently attached heme. The binding of exogenous ligands displaces the distal histidine and induces a conformational rearrangement involving the reorganization of internal void volumes. The formation of passageways within the resulting conformation is thought to facilitate ligand exchange and play a functional role. Here we monitored the perturbation induced by pressure on the ferric bis-histidine and cyanide-bound states of GlbN using ¹H–¹⁵N HSQC NMR spectroscopy. We inspected the outcome with a statistical analysis of 170 homologous 2/2 hemoglobin sequences. We found that the compression landscape of GlbN, as represented by the variation of an average chemical shift parameter, was highly sensitive to ligand swapping and heme covalent attachment. Stabilization of rare conformers was observed at high pressures and consistent with cavity redistribution upon ligand binding. In all states, the EF loop was found to be exceptionally labile to pressure, suggesting a functional role as a semi-flexible hinge between the adjacent helices. Finally, coevolved clusters presented a common pattern of compensating pressure responses. The high-pressure dissection combined with protein sequence analysis established locations with volumetric signatures relevant to residual communication of 2/2 hemoglobins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Expression patterns and adaptive functional diversity of vertebrate myoglobins

Recent years have witnessed a new round of research on one of the most studied proteins - myoglobin (Mb), the oxygen (O₂) carrier of skeletal and heart muscle. Two major discoveries have stimulated research in this field: 1) that Mb has additional protecting functions, such as the regulation of in vivo levels of the signaling molecule nitric oxide (NO) by scavenging and generating NO during normoxia and hypoxia, respectively; and 2) that Mb in vertebrates (particularly fish) is expressed as tissue-specific isoforms in other tissues than heart and skeletal muscle, such as vessel endothelium, liver and brain, as found in cyprinid fish. Furthermore, Mb has also been found to protect against oxidative stress after hypoxia and reoxygenation and to undergo allosteric, O₂-linked S-nitrosation, as in rainbow trout. Overall, the emerging evidence, particularly from fish species, indicates that Mb fulfills a broader array of physiological functions in a wider range of different tissues than hitherto appreciated. This new knowledge helps to better understand how variations in Mb structure and function may correlate with differences in animals' lifestyles and hypoxia-tolerance. This review integrates old and new results on Mb expression patterns and functional properties amongst vertebrates and discusses how these may relate to adaptive variations in different species. This article is part of a special issue entitled: Oxygen Binding and Sensing Proteins.