Molecular and complex-chemical studies on methemoglobin from Chironomus thummi thummi and various isolated fractions]

1. Six different hemoglobin (Hb) fractions were isolated and characterized from the larvae of Chironomus thummi thummi using column chromatographic procedures. 2. Chromatographic and sedimentation-analytic studies (sedimentation coefficients of 2.0 +/- 0.2 (S)) have shown three Hb fractions to exist basically in a monomeric form. The molecular weight of component M-2 was determined by sedimentation equilibrium technique to be 15,470 +/- 400. The dimeric Hb was found to have sedimentation coefficients of 3.0 +/- 0.1 (S) in the weakly acidic pH region. In alkaline milieu, the reversible dissociation proceeds into the monomeric molecules (S20, W = 1.9 +/- 0.1 (S)). Molecular weights vary between pH 5.7 and 9.8 not only with hydrogen ion concentration, but also with protein concentration in correspondence with a dissociation-association equilibrium consisting of monomers and dimers. 3. For the Hb fraction M-2, a friction ratio of f/fo = 1.03 was calculated, suggesting an almost spherical shape of this protein. In contrast, the dimeric component appears to have a much more asymmetric structure (f/fo = 1.19). 4. The indivdual MetHb fractions bind the ligands: fluoride, imidazole and azide with different affinities.     

Facilitation of Hb S polymerization by the substitution of Glu for Gln at beta 121

In an effort to clarify the role of Glu-beta 121 of Hb S molecules in polymerization, we studied the solubility and kinetics of polymerization of various mixtures of deoxyhemoglobins S (Glu-beta 6----Val) and D Los Angeles (Glu-beta 121----Gln). It is known that patients with Hb S-D Los Angeles have a relatively severe clinical course. Mixtures of Hb S and Hb D Los Angeles polymerized after a distinct delay time, the length of which depended on the initial hemoglobin concentration and the fraction of Hb S in the mixture. There was a linear relationship between the logarithmic plot of delay time and initial hemoglobin concentration. The line for a 1:1 mixture of Hb S and Hb D Los Angeles shifted to the right of that for deoxy-Hb S by 0.08. This shift is much smaller than the shift of 0.32 for 1:1 AS mixtures. From these data, the probability factor for nucleation of S-D Los Angeles hybrid hemoglobin was calculated to be 1.16, which is higher than that of Hb S (1.0) and AS hybrid hemoglobin (0.5). The degree of co-polymerization of Hb D Los Angeles in S-D Los Angeles mixtures was similar to that of Hb A in AS mixtures. The critical concentration for the polymerization of Hb D Los Angeles was between that of Hb A and Hb Machida, which has the same amino acid substitution (Glu----Gln) at the beta 6 position. These results suggest that the protein interaction of Hb S molecules during nucleation involves at least two steps. First, the Val-beta 6 of a Hb S molecule interacts hydrophobically with the Phe-beta 85 and the Leu-beta 88 of an adjacent Hb S molecule. In the second step, Glu-beta 121 weakens the interaction with His-beta 116 and Pro-alpha 114. The substitution of Glu-beta 121----Gln may strengthen this second reaction and facilitate nucleation as well as polymerization.     

Physiological characteristics of human red blood cell ghosts

The properties of ghosts prepared by hypotonic hemolysis at various ratios of cells (C) to hemolyzing solution (H) have been studied. At all ratios, hemoglobin (Hb) was found to be distributed equally between the ghost and supernatant compartments. Techniques employing Fe(59)-labelled Hb showed that during hemolysis all of the Hb is exchangeable and that following hemolysis the ghost is impermeable to Hb. Ghosts containing defined fractions of their original Hb were prepared by appropriately altering the ratio C/H. When washed and suspended in 0.17 M NaCl-PO(4)-buffered media, the ghosts returned to their initial volume, recovered normal shape, and behaved as osmometers. The rate of rehemolysis of these reconstituted ghosts was observed to be proportional to the concentration of Hb in the ghosts. The rate of rehemolysis was accelerated by the addition of n-butyl alcohol (BA). For a given concentration of BA, temperature, and Hb content the rate of rehemolysis was minimal around the isoelectric point of Hb. Rehemolysis by BA was inhibited by the addition of sucrose to the medium. K influx and outflux were measured and found to be increased by the addition of BA and not influenced by the presence of sucrose. These results on the rehemolytic characteristics of ghosts are consistent with and support the colloid-osmotic theory of hemolysis.     

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin was investigated by accurately measuring and analyzing the oxygen equilibrium curves of human adult hemoglobin in the presence and absence of various concentrations of one or two of the following materials: chloride (Cl-), 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). Each equilibrium curve was analyzed according to the Adair equation to evaluate the four-step oxygen equilibrium constants (Adair constants) and the median oxygen pressure. The binding constants of the anions for the molecular species of hemoglobin carrying j oxygen molecules, Hb(O2)j(j=0,1,...,4), were evaluated from the dependences of the Adair constants and the median oxygen pressure on the anion concentration by introducing a model which takes the competitive binding of Cl- and DPG or IHP into account. Assumptions made in the model are: (a) the hemoglobin molecule has two oxygen-linked binding sites for Cl- which are equivalent and independent and (b) no Cl- can be bound to hemoglobin to which DPG or IHP is already bound and vice versa. Thus, we could obtain values for the intrinsic binding constants of Cl- and DPG, i.e., the constants in the absence of other competitive anions. For IHP, only the binding constants and apparent binding constants for Hb and Hb(O2)2 were obtained. Values of the Cl- binding constants and apparent binding constants for DPG and IHP, i.e., the binding constants in the presence of Cl- for Hb and Hb(O2)4, were in reasonable agreement with literature values. From the binding constants we calculated anion binding curves for Hb(O2)j(J=0,1,...,4), the number of anions bound to Hb(O2)J, And the relationship between fractional anion saturation of hemoglobin and fractional oxygen saturation. The numbers of released anions are not uniform with respect to oxygenation step. This non-uniformity is the reason for the changes in the shape of the oxygen equilibrium curve with anion concentration changes and for the non-uniform dependences of the Adair constants on anion concentration, and also results in non-linear relations between anion saturation and oxygen saturation. The anion binding constants and various binding properties of the anions derived from those constants are consistent with those observed by other investigators using different techniques, indicating that the present model describes the oxygen-linked competitive anion binding well.     

"Bridge" structures between nucleic acid molecules fixed in the structure of a liquid crystal

The binding of daunomycin and copper ions to poly(I).poly(C) molecules fixed in a particle of a liquid-crystalline dispersion was studied. A thermodynamic model of adsorption was developed, which makes it possible to describe the formation of complexes of a particular kind, "bridges" that connect adjacent nucleic acid molecules fixed in a liquid crystal. The bridges represent chelate complexes, which incorporate the molecules of the antibiotic daunomycin and copper ions. Equations describing the dependence of the concentration of these bridges in solution on the concentration of their constituents were derived. The family of dependences of experimental amplitudes of bands in CD spectra typical of "bridge" structures on the concentration of copper ions represents a set of S-shaped curves, and, as the concentration of daunomycin in solution increases, the level of saturation of these curves increases. The analysis of experimental data with the use of this model suggests that the structures of this type compete with daunomycin molecules for the binding sites on poly(I).poly(C). By using this model, the energies of formation of bridge structures were calculated.     

Optimization of production and downstream processing of the almond beta-glucosidase-mediated glucosylation of glycerol.

This article describes the synthesis of glyceryl glucoside from glycerol and glucose with almond beta-glucosidase as the catalyst. A yield of 54% (0.45 mmol/g) was obtained. The influence of the enzyme stability, the water concentration, and the water activity on the glucoside yield were determined. A molar fraction-based equilibrium constant of 2.4 +/- 0.6 was found, with which the glucoside yield could be calculated for all possible combinations of initial substrate and water fractions in the reaction mixture. A model was used to optimize the glucoside yield while minimizing one of the substrate concentrations at equilibrium. This straightforward model gives a good prediction of the measured glucoside yield, according to a parity plot.     

Strong and weak binding of water to proteins studied by NMR triple-quantum filtered relaxation spectroscopy of (17)O-water

The triple-quantum filtered (TQF) spin-echo signal of (17)O-water, in the presence of proteins, was analysed to yield estimates of the number of weakly, and strongly bound water molecules. The analysis used a constrained direct iterative regression procedure with a three-state model of fast-exchange. Thus, the population size of free, weakly, and strongly bound water were determined simultaneously. The two fractions of the bound water were estimated by using correlation time(s) estimated in other studies. Bovine serum albumin (BSA), basic pancreatic trypsin inhibitor (BPTI), lysozyme and oxyhaemoglobin were studied. Of the four proteins, BSA contained the largest number of strongly and weakly bound water molecules, there being approximately 30 of the former and approximately 3000 of the latter under conditions of high protein concentration. The correlation time of the proteins increases with their concentration in solution, and when this was taken into account for BSA the estimated number of strongly bound water molecules did not change significantly. This NMR technique, and data analysis, will probably also be useful in studies of water binding and mobility in various systems including hydrogels, protein networks, membranes, cells and tissues.     

The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.     

Kinetics of denaturation of human and chicken hemoglobins in the presence of co-solvents

The stability of four hemoglobins (Hb) in dimer forms (low concentration) were investigated by the kinetics of denaturation. The rate constants of denaturation were obtained by variation of 280 nm absorption versus time in 10 mM Tris-HCl, 10 mM EDTA, pH 8.0 at 45 degrees C in the absence and presence of 0.5 M ethanol, dimethyl sulfoxide (DMSO), formamide, and glycerol. The results show the trend of rate constants in different co-solvents in the following order: chicken hemolysate < human hemolysate and chicken Hb D < chicken Hb A. The buried surface area was calculated for Hb samples in the absence of co-solvents. Accordingly, the trend points out that: chicken Hb D > chicken Hb A > human Hb A. These results suggest that both chicken hemolysate and chicken Hb D are relatively more stable than human and chicken Hb A, respectively. However, the denaturation rate constants of Hb in different co-solvents have designated the following order: ethanol > DMSO > formamide > glycerol. As a matter of fact, this phenomenon is an indication of an increase in the denaturation capacity (DC) and hydrophobicity, and a decrease in the surface tension of the solution in the preceding co-solvents.     

Experimental data and modelling of apparent molar volumes, isentropic compressibilities and refractive indices in aqueous solutions of glycine + NaCl

Experiments have been performed at 298.15 K to measure the density, sound velocity and refractive index of glycine in aqueous solutions of NaCl over a wide range of both glycine and NaCl concentrations. The values of apparent molar volume and isentropic compressibility of glycine were calculated from the measured data. The results show a positive transfer volume of glycine from an NaCl solution to a more concentrated NaCl solution. This indicates that the size of a glycine molecule is larger in a solution with higher NaCl concentration. The negative values of apparent isentropic compressibility imply that the water molecules around the glycine molecules are less compressible than the water molecules in the bulk solution. These effects are attributed to the doubly charged behaviour of glycine and to the formation of physically bonded ion-pairs between the charged groups of glycine and sodium and chloride ions. The formation of ion-pairs, whose extents of binding reactions depend on the concentrations of both NaCl and glycine, alter the hydration number of glycine. This also explains the reason for the increase in the size of glycine with an increase in the NaCl concentration. A model based on the Pitzer formalism has been developed to correlate the activity coefficient, apparent molar volume and isentropic compressibility of glycine in aqueous solutions of NaCl. The results show that the model can accurately correlate the interactions in aqueous solutions of glycine and NaCl.     

Hemoglobin polymers during their circulation in the vascular bed of animals

The effect of molecular mass of 1 g/kg hemoglobin (Hb) modified by glutaraldehyde and pyridoxal-5'-phosphate on the duration of Hb presence in the rabbit circulation was studied. Hb concentration and met-form content were measured in plasma samples taken 5 min, 6, 18 and 24 h after the injection. Gel and ion exchange chromatography was performed. Predominant clearance of low-molecular fractions within the first 6 h of circulation was shown, it was followed by a rise in high-molecular fraction (greater than 300,000) content, that might be caused by the interaction of modified Hb with plasma proteins. This corroborates the decrease in the overall charge of the circulating fractions.     

Oxygen binding and other physical properties of human hemoglobin made in yeast.

Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cation and anion of an electrolyte on apparent molar volume, isentropic compressibility and refractive index of glycine in aqueous solutions

Experiments at 298.15 K have been performed to measure the density, velocity of sound and refractive index in three water+glycine+electrolyte systems. The electrolytes studied were KCl, KNO3 and NaNO3. The values of apparent molar volume and isentropic compressibility of glycine in aqueous electrolyte solutions were calculated from the measured data. The results obtained in this study and those reported previously for water+glycine+NaCl system have been comparatively studied. The results show that the nature of both the cation and the anion of an electrolyte influence the behaviour of glycine in aqueous solutions. For all four electrolytes studied, the comparison shows a positive volume transfer for glycine from an electrolyte solution to a more concentrated solution of the same electrolyte. The results also show a negative apparent isentropic compressibility for glycine in the presence of the electrolytes studied. These effects indicate that the volume of a glycine molecule is larger in solutions with higher electrolyte concentration and the water molecules around the glycine molecules are less compressible than the water molecules in the bulk solution. These effects were attributed to the doubly charged behaviour of glycine and to the formation of physically bonded ion-pairs between the charged groups of glycine and the cation and the anion of the electrolyte.     

Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains

A murine model of sickle cell disease was tested by studying the polymerization of hybrid hemoglobin tetramers between alpha mouse and human beta S or beta S Antilles chains were prepared from Hb S Antilles, which was a new sickling hemoglobin inducing a sickle cell syndrome more severe than Hb S. The hybrid molecules did not polymerize in solution, indicating that the mouse alpha chains inhibited fiber formation. Consequently, a mouse model for sickle cell disease requires the transfer and expression of both alpha and beta S or beta S Antilles genes.     

Dynamics of anaerobic growth of Saccharomyces cerevisiae in the chemostat.

A kinetic Monod model has been used to describe the dynamic response of a continuous stirred tank fermentor (CSTF) to changes in dilution rate. A general analytical solution of a linearized model was obtained. Experimental results (Vairo et al. 1977) of continuous anaerobic culture of Saccharomyces cerevisiae have verified the model quantitatively. For step disturbances on the dilution rate the responses of biomass concentration and the outlet substrate concentration were calculated on a digital computer and compared with the experimental data.     

Possibility of wide-angle neutron scattering in the study of proteins and nucleic acids in solutions

A method is proposed for calculating wide-angle neutron scattering curves of biopolymers at any fraction of heavy water (D2O) in solution. The method permits to accurately take into account the phenomenon of deuteroexchange. By this method neutron scattering curves of proteins and DNA have been calculated. The calculations have shown that at optimal fractions of D2O in solution the profiles of neutron scattering curves and their sensitivity to conformational rearrangements in protein molecules turned out to differ very little from those of corresponding X-ray curves. Thus the neutron scattering curves do not contain any additional information (as compared with those contained in X-ray scattering curves) on the structure of proteins in solution. On the contrary, neutron and X-ray scattering curves of DNA differ significantly at all fractions of D2O in solution and therefore the methods of wide-angle neutron and X-ray scattering could become mutually complementary in studying the structure of nucleic acids in solution.     

A transport kinetic concept for ion uptake by plants

Summary A previously presented transport kinetic model for ion uptake by plants was tested by using data for copper uptake by barley grown in soil or water culture and corresponding data for copper concentration in the soil solution or culture solution. As no significant differences were obtained between experimentally determined values for copper uptake and those calculated by using the model, it was concluded that the experimental evidence was consistent with the proposed transport kinetic model. The model allows the Michaelis-Menten Constant of ion uptake and the Mean Maximal Rate of ion uptake to be calculated for various time intervals of the growth period of plants. The Michaelis-Menten Constant for copper uptake by barley grown in soil was the same as that for plants grown in water culture, provided that the soil solution and the water culture solution had the same chemical composition. The concentration of copper in the soil solution, at which the rate of copper uptake was zero, was 2–4 times larger than corresponding values obtained in water culture solution. re]19751028     

On the separation ability of various Ficoll gradient solutions in zonal centrifugation

Ficoll samples of various molecular weights, Mr(15-4200) X 10(3), forming gradient solutions with different separation resolutions and a more selective action have been obtained by fractionation of Ficoll 400 polysaccharide. On the basis of sedimentation-diffusion data and viscometry of Ficoll fractions in water and dimethylformamide, conclusions about the type of branching of macromolecules and the intensity of intramolecular hydrodynamic interaction were made; the size and shape asymmetry of Ficoll molecules were calculated in connection with their mobility in solution.     

Novel allosteric conformation of human HB revealed by the hydration and anion effects on O(2) binding.

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O(2) affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the "osmotic stress" method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl(-) and of DPG concentration. We find that only approximately 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with approximately 72 that bind in the presence of more than 50 mM Cl(-) or more than 15 microM DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl(-), DPG, and H(2)O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.     

Preparative fractionation of lignin by gel-permeation chromatography

The combination of an agarose gel (Bio-Gel A) and a dioxane–water (1:1) solvent system allowed the fractionation, on a preparative scale, of a very polydisperse, non-derivatized lignin preparation (enzymatically liberated lignin prepared from sweetgum sapwood with Lenzites trabea). Three fractions differing markedly in molecular weight were obtained. A gel of crosslinked alkylated dextran (Sephadex LH-20) with the same solvent system allowed division of the lowest molecular weight fraction into two fractions. These materials were characterized by measurements of intrinsic viscosity and number-average molecular weights in dimethylformamide and dioxane–water. It was established that the two highest molecular weight fractions were associated in an average trimeric form in dioxane-water (1:1) as compared to the form (considered to be molecular) that occurred in dimethylformamide. Molecular size distributions and eluant volumes of the fractions were determined with a Sephadex G-100–formamide system, the latter being one of the most powerful nonaqueous solvents for lignin. Adsorption effects were known to be absent in this case, and the lignin molecules were considered to be unassociated in formamide. The four fractions were distinguishable with the formamide–G-100 system, thus indicating that the original fractionation was based on molecular size. The enzymatically liberated lignin contained molecules that comprised a continuum of molecular weights from approximately monomeric to molecules that were at the limit of the solvating power of dioxane–water (1:1) and dimethylformamide. Limited physicochemical data were consistent with a compact, approximately spherically symmetric shape of the lignin in solution.