Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential

On-line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb-synthesizing E. coli strain and the iso-genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8-fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen-limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb-expressing cells responds to rapidly changing oxygen tension, which is common in large-scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.(c) John Wiley & Sons, Inc.     

Protein fractionation using electrostatic interactions in membranel filtration

One of the critical factors limiting the development of membrane systems for protein fractionation has been the poor selectivity that has generally been obtained with these membrane devices. We have demonstrated that it is possible to dramatically improve the selectivity of available membrane systems by exploiting the different electrostatic interactions between the two proteins and the membrane. The separation factor for the albumin-hemoglobin system could be increased to more than 70 simply by reducing the salt concentration and adjusting the pH to around 7 (near the isoelectric point of hemoglobin). This very high selectivity was a direct result of the strong electrostatic exclusion of the charged albumin from the membrane pores under these conditions. This high selectivity makes it possible to very effectively separate these albumin-hemoglobin mixtures using membrane filtration, and this was demonstrated experimentally using both a simple batch filtration process and a continuous diafiltration system. The hemoglobin recovery in the diafiltration experiment was greater than 70% after a 3-diavolume filtration, with the Hb purification factor being around 100 under these conditions. These results clearly demonstrate the potential of membrane systems for the fractionation of proteins even with very similar molecular weights. (c) 1995 John Wiley & Sons, Inc.     

Myoglobin discriminates between O2, NO, and CO by electrostatic interactions with the bound ligand

 Most biological substrates have distinctive sizes, shapes, and charge distributions which can be recognized specifically by proteins. In contrast, myoglobin must discriminate between the diatomic gases O₂, CO, and NO which are apolar and virtually the same size. Selectivity occurs at the level of the covalent Fe-ligand complexes, which exhibit markedly different bond strengths and electrostatic properties. By pulling a water molecule into the distal pocket, His64(E7)¹ inhibits the binding of all three ligands by a factor of ∼10 compared to that observed for protoheme-imidazole complexes in organic solvents. In the case of O₂ binding, this unfavorable effect is overcome by the formation of a strong hydrogen bond between His64(E7) and the highly polar FeO₂ complex. This favorable electrostatic interaction stabilizes the bound O₂ by a factor of ∼1000, and the net result is a 100-fold increase in overall affinity compared to model hemes or mutants with an apolar residue at position 64. Electrostatic interaction between FeCO and His64 is very weak, resulting in only a two- to three-fold stabilization of the bound state. In this case, the inhibitory effect of distal pocket water dominates, and a net fivefold reduction in K _CO is observed for the wild-type protein compared to mutants with an apolar residue at position 64. Bound NO is stabilized ∼tenfold by hydrogen bonding to His64. This favorable interaction with FeNO exactly compensates for the tenfold inhibition due to the presence of distal pocket water, and the net result is little change in K _NO when the distal histidine is replaced with apolar residues. Thus, it is the polarity of His64 which allows discrimination between the diatomic gases. Direct steric hindrance by this residue plays a minor role as judged by: (1) the independence of K _O2, K _CO, and K _NO on the size of apolar residues inserted at position 64, and (2) the observation of small decreases, not increases, in CO affinity when the mobility of the His64 side chain is increased. Val68(E11) does appear to hinder selectively the binding of CO. However, the extent is no more than a factor of 2–5, and much smaller than electrostatic stabilization of bound O₂ by the distal histidine. Received, accepted: 23 May 1997     

Dynamic properties of some β-chain mutant hemoglobins

The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some β-chain mutant hemoglobins is studied in the temperature range 300–10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the overall bandwidth are singled out With this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wildtype and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme–CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the “polar isosteric” βVal-67(Ell) →Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue βLys-144(HC1) at the C-terminal and residue βCys-112(G14) at the α₁β₁ interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the β-N-terminal and at the α₁β₂ interface have no effect on the dynamic properties of the heme pocket. © 1995 Wiley-Liss, Inc.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies

The thermal induction of chemiluminescence of luminol-horseradish peroxidase-labeled erythrocytes from pigs, rats, and man was studied. The luminescent responses of rat, porcine, and human erythrocytes to heating were linear in respect to logs of counts per minute versus temperature. Landrace-Duroc crossbred pigs with a history of malignant hyperthermia (porcine stress syndrome) and Poland-China-miniature pigs inbred for malignant hyperthermia (MH) yielded erythrocytes with high-level thermochemiluminescence (TCL). Sprague-Dawley rat erythrocytes were intermediate in their production of TCL. Normal human and MH-resistant miniature swine erythrocytes produced low-level TCL. However, pretreatment of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in high-level TCL. Furthermore, halothane enhanced the TCL of CDNB-treated human erythrocytes and Landrace-Duroc porcine erythrocytes that were not treated with CDNB. Red blood cells from pigs susceptible to the porcine stress syndrome demonstrated a TCL response very similar to CDNB-treated erythrocytes.     

Mutant hemoglobin stability depends upon location and nature of single point mutation

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.     

Inhibition of heme synthesis in bone marrow cells by succinylacetone: effect on globin synthesis

The effects of 4,6-dioxoheptanoic acid (succinylacetone, SA), an inhibitor of delta-aminolevulinic acid dehydratase, on total iron uptake, heme synthesis, and globin synthesis were studied in rat marrow cells in culture in order to examine the coordination of heme and globin synthesis. SA inhibited heme synthesis in both control and erythropoietin-stimulated cells in a dose-dependent fashion; at 10(-3) M, inhibition was complete, whereas at 10(-7) M, there was no significant effect. Inhibition of total iron uptake was also dose-dependent although, at 10(-3) M, it was not complete. The inhibition of heme synthesis by SA was partially overcome by addition of 10(-4) M porphobilinogen or protoporphyrin IX. SA caused an almost complete suppression of globin formation in both erythropoietin-stimulated and unstimulated cells as early as five hours after the addition of the inhibitor. When inhibition of heme synthesis was incomplete, globin synthesis was partially inhibited. These results indicate that heme synthesis is required for erythropoietin-mediated induction of globin synthesis in cultured bone marrow cells.     

Inhibition of invitro sickling by liposome-mediated transport of amino acids into intact human red blood cells

To investigate the role of phenylalanine and tryptophane as potential antisickling agents in intact human SS-red blood cells a liposomal transport system was employed to transfer phenyl-alanine or tryptophane into intact SS-red blood cells. Aromatic amino acids and short peptides containing phenylalanine have been demonstrated to increase the minimum gelling concentration and solubility of deoxy-hemoglobin S in aqueous solution. However, these compounds do not cross the red blood cell membrane under usual incubation conditions. Incorporation of phenylalanine or tryptophane into intact SS-red blood cells $\text{via}$ liposomal transport system markedly inhibited the $\text{in}\text{vitro}$ sickling of deoxy-hemoglobin S. These findings raise the possibility that a nontoxic liposomal transport system which facilitates incorporation of antisickling agents into intact SS-RBC may have significant therapeutic implications in the treatment of sickle cell disease.     

Study on the dehydrating effect of the red cell Na+/K+-pump in nystatin-treated cells with varying Na+ and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na⁺ concentrations and water content. Such cells provided an experimental model to study the effect of Na⁺/K⁺ pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na⁺/K⁺ pump by elevated intracellular Na⁺ caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na_out⁺ : 2 K_in⁺ stoichiometry of the Na⁺/K⁺ pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.