The solubility of sickle and non-sickle hemoglobins in concentrated phosphate buffer.

A new turbidimetric method for the direct measurement of the solubility of oxy- and deoxyhemoglobins (Hb) in concentrated phosphate buffer has been established. The principle of the method is the formation of a homogeneous emulsion when hemoglobin is introduced in concentrated phosphate buffer. The solubility of the oxy and deoxy forms of Hb A, Hb S, Hb C, Hb F, and Hb CHarlem (beta 6Glu leads to Val, beta 73Asp leads to Asn) has been studied. The solubility of deoxy-Hb S was the lowest and the solubility curve was broader than those of the other hemoglobins indicating that the aggregates of deoxy Hb S require more water to be dissolved. The solubility of oxy- and deoxyhemoglobins depends on temperature and pH. The solubility of hemoglobins is increased as the temperature is lowered and the pH is raised. The pH dependency of the solubility of deoxy-Hb S in high phosphate buffer was opposite to that of the minimum gelling concentration of deoxy-Hb S. The order of the solubility of Hb CHarlem, Hb FS, Hb AS, Hb CS, and Hb S in concentrated phosphate buffer corresponds to the order of minimum gelling concentration of these hemoglobins or hemoglobin mixtures. Solubility studies of a 1:1 mixture of deoxy-Hb A and deoxy-Hb S show that deoxy-Hb A aggregates in 2.42 M phosphate buffer in which pure deoxy-Hb A is totally soluble. This result indicates that deoxy-Hb S interacts with deoxy-Hb A and decreases its solubility.     

The effect of organic compounds on the crystal habit and crystallization of normal and sickle cell hemoglobin in phosphate buffer

Comparative studies were made on the effect of numerous organic compounds in promoting the crystallization of human hemoglobin in 1.9 m phosphate, pH 7.0. It was found that alicyclic or benzenoid structures are essential for promoting crystallization of hemoglobin under these conditions. Hemoglobin crystals prepared in the presence of toluene differed in habit from crystals prepared in its absence. It is suggested that steric factors determine the effectiveness of organic substances in promoting the crystallization of hemoglobin and that the heme group is the binding site involved in the complex formation.The solubility of homozygous sickle cell hemoglobin HbS was found to be less than the heterozygous hemoglobins AS and AC or normal hemoglobin HbA in the presence of organic substances promoting the crystallization of hemoglobin.     

Fractionation of Chromatin by thermal precipitation in phosphate buffer.

The stability of sonicated rat liver chromatin in sodium phosphate buffer, pH 6.8 was studied as a function of buffer concentration (0.012 to 0.16 m) and temperature (20 to 98 °C). It was found that as the temperature was increased a stepwise precipitation of chromatin took place which was revealed by the presence of three plateaux (20 to 50 °C, 70 to 75 °C and above 90 °C) and two transitional zones (55 to 70 °C and 75 to 90 °C) on the A₃₂₀ curves and on the percentage precipitated nucleoprotein versus temperature curves as well.This permitted the fractionation of chromatin in 0.08 m-phosphate buffer into three fractions by a stepwise heating at 50 °C (50 °C-pellet) and 98 °C (50–98 °C-pellet and post 98 °C-supernatant). DNA isolated from these fractions was characterized in respect to sedimentation velocity and hybridization with heterogeneous nuclear RNA. The hybridization studies showed a different ability of these three DNA preparations in binding nuclear heterogeneous RNA: 16%, 8% and 30% for DNA isolated from 50 °C-pellet, 50–98 °C-pellet and post 98 °C-supernatant, respectively. The results are discussed in terms of chromatin structure and function.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.     

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.     

Emergence of adaptability to time delay in bipedal locomotion

Based on neurophysiological evidence, theoretical studies have shown that locomotion is generated by mutual entrainment between the oscillatory activities of central pattern generators (CPGs) and body motion. However, it has also been shown that the time delay in the sensorimotor loop can destabilize mutual entrainment and result in the failure to walk. In this study, a new mechanism called flexible-phase locking is proposed to overcome the time delay. It is realized by employing the Bonhoeffer–Van der Pol formalism – well known as a physiologically faithful neuronal model – for neurons in the CPG. The formalism states that neurons modulate their phase according to the delay so that mutual entrainment is stabilized. Flexible-phase locking derives from the phase dynamics related to an asymptotically stable limit cycle of the neuron. The effectiveness of the mechanism is verified by computer simulations of a bipedal locomotion model.     

Molecular properties of R-phycocyanin subunits from Polysiphonia urceolata in potassium phosphate buffer.

The two subunits of R-phycocyanin from Polysiphonia urceolata were isolated and renatured. The renatured subunits were characterized by electrophoresis, molecular weights and spectra. The blue-shifted spectra, fluorescence recovery and restoring of the energy transfer suggested correct refolding of the subunits. The molecular properties of the subunits in potassium phosphate buffer (KPB) were investigated in detail. The total fluorescence yields (Q(T)) of the beta subunit declined while the energy transfer efficiency (E(T)) in the beta subunit was promoted with the increase of KPB concentration. On the other hand, both Q(T) and E(T) were enhanced with the increasing of the subunit concentrations. Based on the structural information, the fluorescence quenching in high concentrations of KPB was ascribed to less rigid chromophores caused by the weakening of the hydrogen-bond interaction network, while the enhancement of the fluorescence and E(T) was due to the aggregation of the subunits in the ionic solvent. Aggregation was confirmed by cysteine-assisted promotion of renaturation yield and stability, as well as equilibrium unfolding tests. Optimal conditions were proposed for the refolding/unfolding studies, under which the subunits were mainly monomeric. Compared to that in C-PC, the blue-shifted spectrum of PCB in R-PC is suggested to bring larger energy transfer efficiency, probably due to the necessity of the light harvesting for P. urceolata living in deep water.     

Oxidation of low-density lipoprotein by copper and iron in phosphate buffer

We examined the effect of phosphate buffer on the iron- and copper-catalyzed peroxidation of low-density lipoprotein (LDL). The incubation of LDL with CuSO4 in 0.15 M NaCl led to the peroxidation of LDL as evidenced by the detection of thiobarbituric acid-reactive substances (TBARS) and lipid hydroperoxides (LPO). The peroxidation of LDL was also observed with FeSO4 and FeCl3 in 0.15 M NaCl, although there was a lag phase with FeCl3. In 10 mM phosphate buffer, the peroxidation of LDL was observed with CuSO4 to an extent similar to that in 0.15 M NaCl. However, the peroxidation induced by incubation with FeSO4 and FeCl3 was significantly inhibited in phosphate buffer. Iron and copper each formed a complex with lipoprotein during incubation with LDL in 0.15 M NaCl. Although no effect on the formation of copper-LDL complex was observed in phosphate buffer, the formation of iron-LDL complex was reduced in the buffer. These observations suggest there are marked differences in the peroxidation of LDL and in the formation of complexes with LDL between iron and copper in phosphate buffer.     

Aggregation of normal and sickle hemoglobin in high concentration phosphate buffer

Sickle cell disease is caused by a mutant form of hemoglobin, hemoglobin S, that polymerizes under hypoxic conditions. The extent and mechanism of polymerization are thus the subject of many studies of the pathophysiology of the disease and potential treatment strategies. To facilitate such studies, a model system using high concentration phosphate buffer (1.5 M-1.8 M) has been developed. To properly interpret results from studies using this model it is important to understand the similarities and differences in hemoglobin S polymerization in the model compared to polymerization under physiological conditions. In this article, we show that hemoglobin S and normal adult hemoglobin, hemoglobin A, aggregate in high concentration phosphate buffer even when the concentration of hemoglobin is below the solubility defined for polymerization. This phenomenon was not observed using 0.05 M phosphate buffer or in another model system we studied that uses dextran to enhance polymerization. We have used static light scattering, dynamic light scattering, and differential interference contrast microscopy to confirm aggregation of deoxygenated and oxygenated hemoglobins below their solubility and have shown that this aggregation is not observable using turbidity measurements, a common technique for assessing polymerization. We have also shown that the aggregation increases with increasing temperature in the range of 15 degrees -37 degrees C and that it increases as the concentration of phosphate increases. These studies contribute to the working knowledge of how to properly apply studies of hemoglobin S polymerization that are conducted using the high phosphate model.     

磷酸缓冲液对植物脂质过氧化的影响及其与亚硫酸伤害的关系

以亚硫酸模式处理不同类型植物 ,从脂质过氧化方面探讨了磷酸缓冲液的防护作用 .结果表明 ,植物经磷酸缓冲液处理后 ,对亚硫酸伤害具有明显的防护作用 .磷酸缓冲液可稳定细胞膜结构 ,降低MDA含量 ,增强叶绿素———蛋白质的结合度 ,且与低浓度亚硫酸对SOD活性呈协同效应 .磷酸缓冲液的防护作用因植物种类而异 .同一污染环境下 ,MDA含量高 ,叶绿素结合度低者效应显著 .在测试植物中 ,银杏 >连翘 >小麦苗 .     

Lateral facilitation of Hoffmann-reflexes prior to voluntary movement in a choice reaction time task.

Hoffmann reflexes (H reflexes) were elicited from both legs simultaneously in human subjects at varying intervals after a reaction signal (RS) in a binary choice reaction time task. A left light RS required a rapid plantar flexion of the left foot and a right light RS required a similar rapid response of the right foot. A large faciliataion of reflex amplitude occurred only in the muscle involved in the movement (right of left soleus). The timing of the facilitation indicated that a decision about the status of the RS occurred within 200 msec and probably was completed somewhat earlier. Furthermore, the facilitation of the H reflexes was shown to be closely linked with the organization required for the contractions of the responding muscle. The results are considered in the light of hypothesized mechanisms regulating voluntary movement.     

Sources of conductance changes during bacterial reduction of trimethylamine oxide to trimethylammonium in phosphate buffer

The sources of conductance changes during reduction of trimethylamine oxide to trimethylamine by Escherichia coli with formate as electron donor and in the presence of phosphate buffer were investigated. Theoretical considerations and experimental results suggest that the major source of conductance change is the conversion of dihydrogen phosphate to hydrogen phosphate. This transformation contributes almost twice as much to the total conductance change as does the conversion of uncharged trimethylamine oxide to charged trimethylammonium.     

Temperature and domain size dependence of sickle cell hemoglobin polymer melting in high concentration phosphate buffer.

Deoxygenated sickle cell hemoglobin (Hb S) in 1.8 M phosphate buffer, and carbon monoxide (CO) saturated buffer were rapidly mixed using a stopped-flow apparatus. The binding of the CO to the Hb S polymers and the polymer melting was measured by time resolved optical spectroscopy. Polymer melting was associated with decreased turbidity, and CO binding to deoxy-Hb S was monitored by observation of changes in the absorption profile. The reaction temperature was varied from 20 degrees C to 35 degrees C. Polymer domain size at 20 degrees C was also varied. The data for mixtures involving normal adult hemoglobin (Hb A) fit well to a single exponential process whereas it was necessary to include a second process when fitting data involving Hb S. The overall Hb S-CO reaction rate decreased with increasing temperature from 20 degrees C to 35 degrees C, and increased with decreasing domain size. In comparison, Hb A-CO reaction rates increased uniformly with increasing temperature. Two competing reaction channels in the Hb S-CO reaction are proposed, one involving CO binding directly to the polymer and the other involving CO only binding to Hb molecules in the solution phase. The temperature dependence of the contribution of each pathway is discussed.