The interaction of inositol hexaphosphate with methaemoglobin

1. Inositol hexaphosphate causes the shape of the oxidation-reduction equilibrium curve to become hyberbolic at acid pH values. 2. Inositol hexaphosphate also causes a decrease in the alkaline oxidation Bohr effect at these same pH values. 3. These results support the idea that inositol hexaphosphate causes methaemoglobin to take up the deoxyhaemoglobin quaternary structure at pH6.5.     

HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.     

Mixed gelation theory. Kinetics, equilibrium and gel incorporation in sickle hemoglobin mixtures.

This paper outlines a theoretical formalism for describing the gelling behavior of sickle cell hemoglobin in mixtures with other hemoglobin and non-hemoglobin proteins. Experimental applications are reported for hybridized and unhybridized mixtures of HbS (sickle hemoglobin), HbA (adult hemoglobin), HbF (fetal hemoglobin), and HbC Harlem. The theory is a general one based on a modification of the sol—gel phase equilibrium equation to take into account the varying tendencies of different hemoglobin species to promote gelation, and specific hemoglobin interactions are encoded in gelling coefficients which quantify gelling capability. Gelling coefficients for the hemoglobin species dealt with here are evaluated by measuring incorporation into the polymer phase in S-A, S-F, and S-C_H mixtures. Given this information, the theory is found to provide accurate prodictions for the equilibrium gelling behavior of the calibrating pairs themselves when they are hybridized or unhybridized, for gelation kinetics in diverse mixtures of these species taken two, three and four at a time, for the anomalous equilibrium and kinetic gelling behavior of A- C_H mixtures, and it also accounts for a variety of results previously published by others. Apparently, given the gelling coefficients for any mutant hemoglobin, one can compute gelling behavior (equilibrium, kinetics, incorporation, etc.) in any specified mixture with any other known hemoglobin(s). The gelling coefficients for any mutant hemoglobin depend upon, and therefore provide information about, gel interactions at the mutant site. From the gelling coefficients one can also obtain the change in free energy of interaction in the gel due to the altered residue. Experimental approaches are described which allow an analysis for the gelling coefficients of any mutant hemoglobin to be performed in a few hours.     

Conversion of oxyhaemoglobin into methaemoglobin by ferricytochrome b5.

Ferricytochrome b5 was found to convert oxyhaemoglobin into methaemoglobin under conditions previously found to be optimal for complex-formation between ferricytochrome b5 and methaemoglobin [Mauk & Mauk (1982) Biochemistry 21, 4730-4734]. As this reaction is completely inhibited by CO, it is proposed that oxyhaemoglobin is oxidized after O2 dissociation, as has been suggested for the oxidation of oxyhaemoglobin by inorganic complexes. From the present analysis, ferricytochrome b5 seems unlikely to contribute significantly to methaemoglobin formation in vivo. Nevertheless, this observation provides a relatively convenient means of investigating the mechanism by which these two proteins interact.     

Characterization of the interactions of NADH with the dimeric and tetrameric states of methaemoglobin

The binding of NADH to the dimeric (αβ) and tetrameric (α₂β₂) states of human aquomethaemoglobin has been characterized by sedimentation equilibrium studies of the effect of the concentration of free ligand on the macromolecular state of the haemoprotein. Both macromolecular states of aquomethaemoglobin exhibit a single binding site for NADH, which interacts approximately tenfold more strongly (6000 cf. 700 M⁻¹) with the tetramer under the conditions studied (pH 6.0, I 0.10). Because the structure of aquomethaemoglobin resembles that of the deoxy state of haemoglobin, there is a high probability that organic phosphates also bind to dimeric deoxyhaemoglobin, a phenomenon which is not considered in thermodynamic treatments of the interplay between oxygen binding and its allosteric inhibition by 2,3-bisphosphoglycerate. Fortunately, the equilibrium constant for deoxyhaemoglobin self-association is so large that neglect of the interaction between allosteric inhibitor and dimeric haemoglobin is an oversight that should have no deleterious implications in the resultant thermodynamic analysis of the interplay between the preferential interactions of oxygen and organic phosphate with the various macromolecular states of deoxyhaemoglobin.     

Calcium-sensitive, actin-related gelation of cell extracts of thermosensitive Chinese hamster lung cells capable of anchorage-independent growth.

The extent of actin-related gelation of extracts of thermosensitive Chinese hamster lung (CHL) cells capable of anchorage-independent growth was studied quantitatively by monitoring the total protein in the gel obtained by low-speed centrifugation. The gelation depended on the presence of ATP, KCl, MgCl2, and a reducing agent. Micromolar concentrations of Ca2+ and low doses of cytochalasin B inhibited the actin-related gelation of these extracts. The gelation was more sensitive to inhibition by Ca2+ and cytochalasin B when the extracts were prepared from cells cultured at the permissive rather than the nonpermissive temperature. When various ts mutants were examined, the half-maximal inhibitory dose (HMID) of Ca2+ and cytochalasin B for gelation of extracts of cells cultured at the nonpermissive temperature was between 1.25 and 2.19 times higher than that for extracts of the same cells cultured at the permissive temperature. The values of the HMID for Ca2+ and cytochalasin B changed shortly after the shift in temperature of cell cultures from the nonpermissive to permissive temperature. When cell extracts were incubated at the permissive temperature in vitro for only 15 minutes, these changes in values of HMID were also observed. Analysis of polypeptides of cell extracts and gel pellets on polyacrylamide gel electrophoresis suggested that the decrease in amount of a high-molecular-weight actin-binding protein (250 kDa) may play an essential role in the increased sensitivity to inhibition by Ca2+ and cytochalasin B of actin-related gelation in extracts of these ts mutants.     

Reactions of Adriamycin with haemoglobin. Superoxide dismutase indirectly inhibits reactions of the Adriamycin semiquinone.

The Adriamycin semiquinone produced by the reaction of xanthine oxidase and xanthine with Adriamycin has been shown to reduce both methaemoglobin and cytochrome c. In air, but not N2, both reactions were inhibited by superoxide dismutase. With cytochrome c, superoxide formed by the rapid reaction of the semiquinone with O2, was responsible for the reduction. However, even in air, methaemoglobin was reduced directly by the Adriamycin semiquinone. Superoxide dismutase inhibited this reaction by removing superoxide and hence the semiquinone by displacing the equilibrium: Semiquinone + O2 in equilibrium or formed from quinone + O2-. to the right. This ability to inhibit indirectly reactions of the semiquinone could have wider implications for the protection given by superoxide dismutase against the cytotoxicity of Adriamycin. Oxidation of haemoglobin by Adriamycin has been shown to be initiated by a reversible reaction between the drug and oxyhaemoglobin, producing methaemoglobin and the Adriamycin semiquinone. Reaction of the semiquinone with O2 gives superoxide and H2O2, which can also react with haemoglobin. Catalase, by preventing this reaction of H2O2, inhibits oxidation of oxyhaemoglobin. Superoxide dismutase, however, accelerates oxidation, by inhibiting the reaction of the semiquinone with methaemoglobin by the mechanism described above. Although superoxide dismutase has a detrimental effect on haemoglobin oxidation, it may protect the red cell against more damaging reactions of the Adriamycin semiquinone.     

Effects of pH, 2,3-diphosphoglycerate and salts on gelation of sickle cell deoxyhemoglobin

Gelation of fully deoxygenated sickle cell hemoglobin was assayed by (1) determination of the temperature at which viscosity increased sharply and (2) a high-speed sedimentation equilibrium method in which three zones are seen. These are a pre-gelation zone, a narrow transition zone exhibiting aggregation, followed by a phase change and a zone of gelation. Only the first zone is seen with deoxyhemoglobin A and CO hemoglobins A and S up to about 0·35 g protein/ml. Minimal gelling temperatures by the viscosity method and, by ultracentrifugation, minimal gelling concentrations determined at the onset of aggregation and at the phase change showed: (a) lowering the pH toward 6·7 favors gelation; (b) deoxyhemoglobin S gels more readily in 6 mm-2,3-diphosphoglycerate than in its total absence; (c) 1 m-NaCl and l m-KCl inhibit gelation. The known favoring of gelation by warming is confirmed by the equilibrium method and is about 2% change in minimal gelling concentration per degree.The effects of pH and high ionic strengths are consistent with contributions of specific polar interactions to gel structure. The effect of 2,3-diphosphoglycerate probably depends on known structural changes which this cofactor induces rather than on alteration of the allosteric quaternary structure equilibrium.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue beta-93.

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.     

Rheology and Confocal Reflectance Microscopy as Probes of Mechanical Properties and Structure during Collagen and Collagen/Hyaluronan Self-Assembly

In this work, the gelation of three-dimensional collagen and collagen/hyaluronan (HA) composites is studied by time sweep rheology and time lapse confocal reflectance microscopy (CRM). To investigate the complementary nature of these techniques, first collagen gel formation is investigated at concentrations of 0.5, 1.0, and 1.5 mg/mL at 37°C and 32°C. The following parameters are used to describe the self-assembly process in all gels: the crossover time (t_c), the slope of the growth phase (k_g), and the arrest time (t_a). The first two measures are determined by rheology, and the third by CRM. A frequency-independent rheological measure of gelation, t_g, is also measured at 37°C. However, this quantity cannot be straightforwardly determined for gels formed at 32°C, indicating that percolation theory does not fully capture the dynamics of collagen network formation. The effects of collagen concentration and gelation temperature on k_g, t_c, and t_a as well as on the mechanical properties and structure of these gels both during gelation and at equilibrium are elucidated. Composite collagen/HA gels are also prepared, and their properties are monitored at equilibrium and during gelation at 37°C and 32°C. We show that addition of HA subtly alters mechanical properties and structure of these systems both during the gelation process and at equilibrium. This occurs in a temperature-dependent manner, with the ratio of HA deposited on collagen fibers versus that distributed homogeneously between fibers increasing with decreasing gelation temperature. In addition to providing information on collagen and collagen/HA structure and mechanical properties during gelation, this work shows new ways in which rheology and microscopy can be used complementarily to reveal details of gelation processes.     

Thermoreversible gelation of aqueous mixtures of pectin and chitosan. Rheology

The synergistic interaction between pectin and chitosan in aqueous acid solution and in the gel phase has been studied by oscillatory shear measurements. Mixtures of pectin and chitosan form thermoreversible gels over a broad composition range by lowering the temperature. The value of the gelation temperature depends on the composition of the mixture, with low values for mixtures with low pectin contents. For incipient gels, a power law can describe the frequency dependence of the complex viscosity, with power law exponents close to -1. The gel evolution of pectin-chitosan mixtures upon a temperature quench below the gel point has been studied. Evidence is provided for a relation between gelation and phase separation in the process of temperature-induced gelation of pectin-chitosan mixtures. A simple model is proposed to rationalize the gelation process in these systems.     

Dissimilar aggregation processes govern precipitation and gelation of human IgM cryoglobulins

Cryoglobulinemia is associated with a range of diseases including rheumatoid arthritis, B-cell malignancies, and chronic viral infections. This "cold-sensitivity" condition is caused by cryoglobulins that precipitate, gel, or occasionally crystallize in the cold. Clinical manifestations vary widely in severity, depending on many factors, including the type of cryoglobulin (monoclonal or mixed immunoglobulins) and the physical nature of the aggregates (precipitate, gel, or crystal). Dynamic light scattering (DLS) was used to examine the cold-induced precipitation or gelation of two human cryoglobulins, namely, Pot IgM and Yvo IgM. The DLS assay was highly reproducible, sensitive, and had low intra-assay variations for both IgM cryoglobulins. Distinct processes were revealed to contribute to precipitation and gelation of cryoglobulins. The precipitation of Pot IgM displayed a rapid transition from solution to solid phases, with a wide distribution of aggregate sizes. In contrast, the gelation of Yvo IgM progressed gradually across a broad temperature range to produce a relatively uniform gel matrix. Initial cryoglobulin concentrations determined the kinetics and critical temperatures for both precipitation and gelation. Moreover, the Yvo IgM was observed to have a distinct relationship between concentrations and mean hydrodynamic diameters or particle sizes. Concentration-dependent effects on particle sizes were present, but not as pronounced for the Pot IgM. Precipitation and gelation of cryoglobulins were also found to be differentially responsive to changes in the aqueous environment. Our results indicate that DLS is a rapid, reliable, and sensitive method for characterizing the nature of disease-associated cryoglobulins.     

Dynamic light scattering and circular dichroism studies on heat-induced gelation of hard-keratin protein aqueous solutions

Animal hairs consist of aggregates of dead cells filled with keratin protein gel. We succeeded in preparing water-soluble hard-keratin proteins and reconstructing the keratin gels by heat-induced disulfide linkages in vitro. Here, the roles of intermolecular hydrophobic interaction and disulfide bonding between the proteins in the gel were discussed. Water-soluble keratin proteins consisting of mixtures of type I ( approximately 48 kDa) and type II ( approximately 61 kDa) were prepared from wool fibers as S-carboxymethyl alanyl disulfide keratin (CMADK). The gelation was achieved by heating an aqueous solution containing at least 0.8 wt % CMADK at 100 degrees C. CMADK solutions with different urea or N-ethylmaleimide concentrations or pH were exposed to dynamic light scattering (DLS) and circular dichroism (CD). DLS clarified the gelation point of CMADK solutions and provided information on the changes in keratin cluster size. DLS suggested two types of gelation mechanism. One was the regenerated chemical disulfide bonding between keratins from CMAD parts of chains. After the gel formed, this bond became important to maintain the gel structure. The other was the physical assembly due to hydrophobic interaction between alpha-helix parts of keratin chains. This hydrophobic assembly also played an important role during gelation. CD confirmed a conformational change in the keratin protein, resulting heat-induced gelation. CD clarified the relationship between keratin protein conformation and gelation, i.e., a rodlike conformation with many alpha-helix structures was necessary to associate keratin chains and form a gel network.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).     

Influence of chain length and polymer concentration on the gelation of (amidated) low-methoxyl pectin induced by calcium

The gelation of low-methoxyl pectin (LMP) induced by addition of Ca2+ was studied by measuring the storage modulus as a function of temperature during cooling. Samples with different molar masses were prepared by mechanical degradation. The effect of the molar mass and the pectin concentration on the gelation properties was investigated. The effect of partial amidation was studied by comparing LMP and partially amidated LMP with the same molar mass and degree of methylation. The results are compared to those from a model developed for Ca2+-induced pectin gelation, and good agreement is found except at low concentrations and low molar masses where the gels are weaker than predicted. At low concentrations intrachain bonding weakens the gel, while the presence of small pectin chains weakens the gel because it neutralizes binding sites on larger chains.     

Gel particles from spray-dried disordered polysaccharides

The formation of gel particles from alginate and ι-carrageenan was studied through a novel pathway of formation via an amorphous spray-dried intermediate. Dried biopolymer particles were suspended in solutions of different Ca²⁺ concentration. Particle size ranges and microscopic observation demonstrated that a range of swelling behaviour could be induced, with lower calcium concentrations resulting in more expanded particles, until a lower limit is reached below which particles initially dissolve. For the same calcium charge stoichiometry, larger swollen gel particles were obtained for alginate than for ι-carrageenan. The ability to produce a range of swollen biopolymer gel particle sizes, on the order of 1–600 μm, is attributed to the balance between gelation and dissolution kinetics, with fast gelation kinetics and slow dissolution promoting production of small gel particles whilst fast dissolution with slow gelation leads to larger gel particles. By controlling the solution environment in which rehydration is carried out, it is therefore possible to produce particles with desired degrees of swelling from a single starting material.     

Cytochalasin B inhibits actin-related gelation of HeLa cell extracts

When the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X- 100 is incubated at 25 degrees C, it gels, and actin and a HMWP are progressively enriched in the extract and in gel isolated from extract. CB (greater than or equal to 0.25 muM) inhibits gelation and specifically lowers the concentrations of actin and the HMWP in the fraction which sediments at 100,000 g after incubation. These results indicate that actin and HMWP are partly disaggregated by cytochalasin treatment, and thus that their aggregation is related gelation. Inasmuch as previous results showed that actin is present and HMWP is enriched in the plasma membrane fraction of HeLa cells, the results also point to a possible relation between plasma membrane-associated gel and in vivo effects of CB.