Study on deoxygenation of polymerized bovine hemoglobin and its effects on the molecular stability / 中国输血杂志

【Objective】 To identify the key factors that regulate the physical vacuum stirring deoxygenation process of bovine hemoglobin and evaluate the stabilities of polyHb solution with different oxygenation degree. 【Methods】 The effects of several factors including gas liquid specific phase boundary area, pH values and polyHb concentrations on deoxygenation process were investigated through monitoring the oxygen saturation of polyHb solution. Furthermore, the MetHb contents of the studied polyHb solutions were measured at regular intervals during the experiments to evaluate the molecular stability of the polyHb through monitoring the oxidation degrees of the experimental Hb solutions under the mimic pasteurevirus inactivation conditions. 【Results】 The results showed that the more gas liquid specific phase boundary area could accelerate deoxygenation rate, the better deoxygenation results could be yielded. On the other hand, lower pH value could promote the deoxygenation process of the polyHb solution due to the decreasing of oxygen affinity of the polyhemoglobin molecules. Furthermore, results of investigation about the Hb concentration presented that Hb concentration had no remarkable effects on the deoxygenation of polyHb. In addition, the results from the stability experiments presented that higher degree of deoxygenation could decrease the oxidation process and thus increase the heat tolerance of the polyhemoglobin. 【Conclusion】 The deoxygenation of polyhemglonbin could improve the stability of the polyhemoglobin and the deoxygenation process could be regulated by changing the gas liquid specific phase boundary area and oxygen affinity. This study could provide reference and preliminary basis for the industrialization and development of HBOCs in the future.

Simplified method for determination of the Oxygen Dissociation Curve (ODC) / Método simplificado para determinar la curva de disociación de oxígeno (cdo)

RESUMEN La afinidad de la hemoglobina (Hb) por oxigeno (O2) es un factor importante que influye en el transporte de este gas, especialmente en hipoxia y en diferentes enfermedades como anemia o fibrosis quística. En la medición de la afinidad se usa la determinación de la curva de disociación Hb:O2. El método presentado para establecer la curva de disociación Hb:O2 (CDO) simplifica los protocolos normalmente utilizados, ya que elimina el requerimiento del equipo específico para equilibrar la sangre con oxígeno en niveles fijos de presión parcial (PO2). Mediante el uso de ecuaciones matemáticas es posible establecer la cinética de saturación de la hemoglobina (SO2) a valores crecientes de PO2. De igual forma, mediante el método se determinan aspectos típicos de la unión Hb: O2 como la dependencia del pH (coeficiente de Bohr) y el tipo de asociación de la proteína con su ligando mediante el diagrama de Hill. En virtud de la simplificación realizada, el método es aplicable en prácticas de laboratorio en población humana y animal, así como en la investigación de diferentes condiciones experimentales.

ABSTRACT The affinity of hemoglobin (Hb) for oxygen (O2) is an important factor influencing the transport of this gas especially in hypoxia and in different diseases such as anemia or cystic fibrosis. By the affinity measurement, the determination of the Hb: O2 dissociation curve is used. The presented method to establish the Hb: O2 oxygen dissociation curve (CDO) simplifies the protocols normally used, since it eliminates the requirement of specific equipment to equilibrate blood with oxygen at fixed levels of oxygen pressure (PO2). By using mathematical equations, it is possible to establish the saturation change of hemoglobin (SO2) at increasing oxygen partial pressure. Similarly, the method determines typical aspects of the Hb: O2 binding as the pH dependence (Bohr coefficient) and the association type of protein with its ligand by the Hill diagram. By this simplification, the method is applicable in laboratory practices in human and animal population, as well as in the investigation of different experimental conditions.

Secondary polycythaemia in a Malay girl with homozygous Hb Tak

Hb Tak is one of more than 200 high affinity haemoglobin variants reported worldwide. It resultsfrom the insertion of two nucleotides (AC) at the termination codon, between codon 146 and codon147 of the beta-globin gene [Beta 147 (+AC)]. Polycythaemia is the main clinical feature althoughaffected carriers are usually asymptomatic and do not require intervention. Several case studies inthis region have reported the co-inheritance of Hb Tak with Hb E, delta beta and beta thalassaemiawith one case of homozygous Hb Tak in a Thai boy. In this case report, a cluster of haemoglobinTak was found in a family of Malay ethnic origin. Cascade family screening was conducted whileinvestigating a 4-year old girl who presented with symptomatic polycythaemia. She had 2 previousHb analysis done, at 7-month and 2-year-old with the diagnosis of possible Hb Q Thailand andHomozygous Hb D, respectively. Both diagnosis did not fit her clinical presentations. She was plethoric,had reduced exercise tolerance as well as cardiomyopathy. Her parents were consanguineouslymarried and later diagnosed as asymptomatic carriers of Hb Tak. Consequently, re-analysis of thegirl’s blood sample revealed a homozygous state of Hb Tak. In conclusion, high oxygen affinityhaemoglobin like Hb Tak should be considered in the investigation of polycythaemic patients withabnormal Hb analyses. In this case, DNA analysis was crucial in determining the correct diagnosis.

Characterization and oxygen binding properties of des-Arg human hemoglobin

The role of chloride in the stabilization of the deoxy conformation of hemoglobin (Hb), the low oxygen affinity state, has been studied in order to identify the nature of this binding. Previous studies have shown that arginines 141α could be involved in the binding of this ion to the protein. Thus, des-Arg Hb, human hemoglobin modified by removal of the α-chain C-terminal residue Arg141α, is a possible model for studies of these interactions. The loss of Arg141α and all the salt bridges in which it participates is associated with subtle structural perturbations of the α-chains, which include an increase in the conformational flexibility and further shift to the oxy state, increasing oxygen affinity. Thus, this Hb has been the target of many studies of structural and functional behavior along with medical applications. In the present study, we describe the biochemical characterization of des-Arg Hb by electrophoresis, high-performance liquid chromatography and mass spectroscopy. The effects of chloride binding on the oxygen affinity and on the cooperativity to des-Arg Hb and to native human hemoglobin, HbA, were measured and compared. We confirm that des-Arg Hb presents high oxygen affinity and low cooperativity in the presence of bound chloride and show that the binding of chloride to des-Arg does not change its functional characteristics as observed with HbA. These results indicate that Arg141α may be involved in the chloride effect on Hb oxygenation. Moreover, they show that these residues contribute to lower Hb oxygen affinity to a level compatible with its biological function.

Amino acid sequence of the fetal chain of yak haemoglobin.

The amino acid sequence of the fetal chain of yak haemoglobin was determined. The sequence is the same as that of the fetal chain of bovine haemoglobin. Phenylalanine is present at position 12 of the helix A in the fetal chain while tryptophan is the amino acid at this position in the β-chain of yak adult haemoglobin. This amino acid replacement may be responsible for the higher oxygen affinity of yak fetal haemoglobin than yak adult haemoglobin.