[4] Preparation of derivatives of ferrous and ferric hemoglobin
Publisher Summary
This chapter describes the preparation of derivatives of ferrous and ferric hemoglobin. The preparation of the different derivatives can often be done directly from the hemolysate, without any further purification. This is justified by the fact that hemoglobin is the major proteic component of the erythrocytic cytoplasm. In other animal species, especially in fish, the hemoglobin composition consists of several components in comparable amounts, and purification of the individual components is necessary. From a strictly methodological point of view, as a general rule, very laborious procedures should be avoided to keep the protein in the native form; this is particularly true in the case of mutant hemoglobins exhibiting a reduced stability. A hemoglobin solution can be stored aerobically in the cold for a few days without appreciable changes of its properties. It is advisable to keep the protein in a concentrated solution and at neutral or slightly alkaline pH. A more prolonged storage of hemoglobin can be obtained in its deoxygenated form. However, there are technical difficulties in keeping the solution completely oxygen free, and this is an absolute requirement because partially saturated hemoglobin is more susceptible to autoxidation than is the fully oxygenated protein. The method of choice for keeping hemoglobin, as well as many other proteins, is rapid freezing and storage in liquid nitrogen.
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A screening method for binding synthetic metallo-complexes to haem proteins
2022, Analytical BiochemistryThe introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV–Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.
Ca<sup>2+</sup> as activator of pseudoperoxidase activity of pigeon, Eurasian woodcock and chicken myoglobins: New features for meat preservation studies
2021, Food ChemistryMyoglobin (Mb), hemeprotein that binds dioxygen in muscle, affects meat colour. Moreover, in presence of peroxides, metMb is a potent oxidant involved in oxidative rancidity in meat. Here, following pigeon Mb purification and primary structure mass spectroscopy characterization, we determined its autoxidation rate and pseudoperoxidase activity with respect to chicken and E. woodcock Mbs. The three Mbs exhibit different autoxidation rates (0.153-h−1 pigeon, 0.194-h−1 chicken and 0.220-h−1 E. woodcock Mbs) and similar specificity constant (9.86x103 M−1s−1 pigeon, 8.81x103 M−1s−1 chicken and 9.90x103 M−1s−1 E. woodcock Mbs), considering their pseudoperoxidase activity. Moreover, for the first time, we detected an increase in pseudoperoxidase activity in presence of Ca2+, particularly at pH 5.8. NMR and CD data indicate that the nonspecific Ca2+ binding induces small local structural rearrangements that in turn slightly reduce pigeon Mb thermal stability. However, considering Ca2+ concentration variations before and post-mortem, this finding must be considered for meat preservation.
Effects of free soluble iron on thermal aggregation of hemoglobin
2021, Biophysical ChemistryAggregation of hemoglobin is implicated in the presentation of diseases like sickle cell disease and thalassemia. Hallmark of the disease being imbalance in the production of globin chains leading to aggregation of excess globin chains and aberrant hemoglobins associated with the disease, broadly categorized as hemoglobinopathy. We have studied thermal aggregation of hemoglobin at 70 °C and pH 6.5 using light scattering, flow cytometry and optical microscopy and tried to investigate effects of few abundant soluble metal ions on such aggregation. Our study indicate that only iron, both in Fe2+ and Fe3+ forms, could inhibit hemoglobin aggregation and the extent of inhibition was 60% in presence of 100 mgL−1 FeCl3. Similar effect was not seen in lysozyme aggregation. Metal ions such as, Cu2+, Zn2+ and Ni2+ also did not have any significant effects on hemoglobin aggregation. Results show this important chaperone like behavior of free iron affecting the kinetics and yield of the aggregation process which could have important consequence in the extent of severity of such hematological diseases.
Methemoglobin determination by multi-component analysis in coho salmon (Oncorhynchus kisutch) possessing unstable hemoglobin
2020, MethodsXHemoglobin derivatives are often quantified in blood to establish cardio-respiratory status and possible causes of impaired oxygen transport. The derivative known as methemoglobin results from oxidation of hemoglobin and is pathologically relevant because it cannot transport oxygen. In species and individuals possessing unstable methemoglobin, methemoglobin formation leads to rapid hemichrome formation and precipitation. Oxidizing reagents in standard methemoglobin analysis techniques therefore prevent accurate quantification of hemoglobin oxidative degradation products in species possessing unstable hemoglobin. In this study, we demonstrated that individual coho salmon (Oncorhynchus kisutch) possess unstable methemoglobin. Because molar absorptivities of coho methemoglobin, hemichrome and carboxyhemoglobin were significantly different from humans, the use of previous standard methods leads to an overestimation of methemoglobin in coho. Spontaneous conversion of methemoglobin to hemichrome was also demonstrated in Chinook (O. tshawytscha), pink (O. gorbuscha) and chum salmon (O. keta), but not steelhead (O. mykiss), indicating there may be a frequent need to account for unstable hemoglobin when quantifying methemoglobin in salmonids.
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Our method builds upon multi-component analysis (MCA) by using a multivariate modeling technique to derive the coho-specific molar absorptivities of major hemoglobin derivatives
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This approach fills a current need for the accurate quantification of methemoglobin in fishes possessing unstable hemoglobin
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Chaperone potential of erythroid spectrin: Effects of hemoglobin interaction, macromolecular crowders, phosphorylation and glycation
2019, Biochimica et Biophysica Acta - Proteins and ProteomicsSpectrin, the major protein component of the erythrocyte membrane skeleton has chaperone like activity and is known to bind membrane phospholipids and hemoglobin. We have probed the chaperone activity of spectrin in presence of hemoglobin and phospholipid SUVs of different compositions to elucidate the effect of phospholipid/hemoglobin binding on chaperone function. It is seen that spectrin displays a preference for hemoglobin over other substrates leading to a decrease in chaperone activity in presence of hemoglobin. A competition is seen to exist between phospholipid binding and chaperone function of spectrin, in a dose dependent manner with the greatest extent of decrease being seen in case of phospholipid vesicles containing aminophospholipids e.g. PS and PE which may have implications in diseases like hereditary spherocytosis where mutation in spectrin is implicated in its detachment from cell membrane. To gain a clearer understanding of the chaperone like activity of spectrin under in-vivo like conditions we have investigated the effect of macromolecular crowders as well as phosphorylation and glycation states on chaperone activity. It is seen that the presence of non-specific, protein and non-protein macromolecular crowders do not appreciably affect chaperone function. Phosphorylation also does not affect the chaperone function unlike glycation which progressively diminishes chaperone activity. We propose a model where chaperone clients adsorb onto spectrin's surface and processes that bind to and occlude these surfaces decrease chaperone activity.