Effect of C02 binding to haemoglobin on the reactivity of the SH groups at position beta 93

In deoxygenated human haemoglobin A_II and sheep haemoglobin B, in the presence of CO₂ the rate of reaction of the SH groups at position ß₉₃ decreased significantly, but did not change in deoxygenated haemoglobin A_Ic, where the N-terminal α amino groups of the ß chains are blocked. In the absence of CO₂ the SH reaction rates were identical for all three haemoglobins in deoxy form, but differed for the respective oxyhaemoglobins. In the presence of CO₂ the individual rate constants for oxyhaemoglobin were not altered. It is concluded that binding of CO₂ to haemoglobin leads primarily to a stabilisation of the tertiary deoxy structure of the individual subunits, rather than to a stabilisation of the deoxy quaternary structure of the tetramer.     

Soret circular dichroism of cobalt-substituted myoglobin and hemoglobin derivatives

Circular dichroic spectra in the Soret region were obtained for the following cobalt-substituted hemoproteins: ^CoMb³, ^CoMbO₂, ^CoMbNO, ^CoMb⁺, ^CoHb, ^CoHbO₂, ^CoHbNO and ^CoHb⁺ and compared with the corresponding spectra of the native species to delineate the sensitivity of Soret circular dichroism to ligation, quaternary structures, metal ion substitution and its magnetic moment. Soret rotational strengths, R, were calculated, and dissymmetry ratios were used to reveal hidden transitions. The results indicate that Soret circular dichroism is sensitive to the metal ion, its oxidation state, ligation and local environment but neither to quaternary structural changes as proposed by Ferrone &; Topp (1975), nor to the magnetic moment of the metal ion as suggested by Li &; Johnson (1969).     

Studies on erythrocruorin. IV. Circular dichroism of earthworm erythrocruorin.

Circular dichroism spectra of Lumbricus erythrocruorin in the absence and in the presence of heme ligands have been analyzed under a variety of experimental conditions in view of the peculiarities in ligand binding displayed by this high molecular weight heme protein (M_r = 3 × 10⁶).The undisaociated molecule exists in a “metastable” form with high cooperativity in oxygen binding, which can be converted into a stable form with low co-operativity either by changes in pH or temperature; circular dichroism spectra of oxyerythrocruorin in the Soret region give direct evidence of a local alteration in the heme environment under the conditions which affect co-operativity in oxygen binding of the undissociated molecule. Similar, although more pronounced changes in the same spectral region are observed in the dissociated molecule of M_r = 270,000, which displays low co-operativity in oxygen binding.Deoxygenation is accompanied by an inversion in the double Soret-Cotton effect, which indicates a substantial rearrangement in the heme environment upon removal of the ligand.The double peak in the Soret region found in all erythrocruorin derivatives can be taken as an indication of a distinctive distribution of the aromatic side-chains interacting with the heme chromophore.     

Binding mode of a cationic porphyrin to parallel and antiparallel thrombin binding aptamer G-quadruplex

Abstract

The spectral properties of meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (TMPyP) in the presence of parallel and antiparallel G-quadruplexes formed from a thrombin-binding aptamer G-quadruplex (5′-G₃T₂G₃TGTG₃T₂G₃) were investigated in this study. Red shift and hypochromism in the Soret absorption band of TMPyP were observed after binding to both parallel and antiparallel G-quadruplexes. The extent of changes in the absorption spectra were similar for both conformers. No circular dichroism spectrum was induced in the Soret region for both parallel and antiparallel G-quadruplexes. This is suggest that there is no or very weak interaction between electric transitions of nucleobases and porphyrin molecule. The accessibility of the neutral quencher I₂ to the G-quadruplex-bound TMPyP was similar for both parallel and antiparallel G-quadruplexes. All these observations suggest that TMPyP was bound at the outside of the quadruplexes, and conceivably interacted with the phosphate group via a weak electrostatic interaction.

Communicated by Ramaswamy H. Sarma     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

Antimicrobial agents from higher plants: Activity and structural revision of flemiflavanone-D from Flemingia stricta

Flemiflavanone-D from Flemingia stricta is active in vitro against Staphylococcus aureus and Mycobacterium smegmatis. Reexamination and interpretation of its spectral properties required revision of its molecular formula to C₂₅H₂₈O₆ and its structure to 2S-5,7,4′-trihydroxy-6-γ,γ-dimethylallyl-3′-γ,γ-dimethylallyl-oxidoflavan-4-one. The new structure was confirmed when reaction with chlorotrimethylsilane sodium iodide in acetonitrile deoxygenated and cyclized flemiflavanone-D to the known dicycloeuchrestaflavanone A. The absolute stereochemistry of flemiflavanone-D was established to be 2S by circular dichroism measurements.     

The solubility of hemoglobin beta 4 S, the mutant subunits of sickle cell hemoglobin

The single subunit hemoglobin β₄^S was found to have a solubility comparable to that of oxygenated rather than deoxygenated Hb S, although it contains twice as many mutant chains as the parent hemoglobin and probably has a quarternary structure similar to deoxyhemoglobin A. This finding supports the assumption that receptor sites in the α chains of sickle hemoglobin are essential for sickling.     

Haemoglobin Bart's hydrops syndrome in Greece.

A case of haemoglobin Bart''s hydrops syndrome was characterised in a Greek family with a history of three other fetuses with hydrops. Family studies showed that both the mother and father carried alpha-thalassaemia genes, and globin-chain synthesis analysis of the present fetus showed a total absence of alpha-chain production. The haemoglobin composition of the fetus was similar to that seen in cases in south-east Asia, and analysis of DNA from the Greek case confirmed the total deletion of the alpha-chain genes. The extent of the deletion, however, differed from that seen in south-east Asian cases and included the loss of one of the embryonic zeta-chain genes. Thus the severe form of alpha-thalassaemia occurs in Greece but has arisen independently from the similar condition in south-east Asia. The condition must be considered in any woman of this racial background who gives a history of unexplained stillbirths.     

Models for ferrous cytochrome b5: Sign inversions in the magnetic circular dichroism spectra of bis-imidazole ferrous porphyrin systems

The magnetic circular dichroism (MCD) spectrum of bis-imidazole ferrous tetraphenylporphyrin in the Soret region is nearly the mirror image of the spectrum of ferrous cytochrome b₅, a bis-imidazole (histidine)-ligated hemoprotein. Based on previous MCD studies of model and protein heme systems, a sign inversion in the spectra of two heme chromophores having essentially the same coordination structure is unexpected. To investigate whether the nature of the porphyrin itself could account for the observed spectral discrepancy, two additional model complexes, bis-imidazole ferrous protoporphyrin IX dimethylester and bis-imidazole ferrous octaethylporphyrin, whose peripheral porphyrin substituent patterns more closely match that of the protein- bound porphyrin, have been prepared and their MCD spectra measured. In these cases, the band pattern of the ferrous protein in the Soret region is successfully reproduced. It therefore appears that the anomalous MCD spectrum of the tetraphenylporphyrin complex can be attributed to the nature and positioning of the peripheral substituents on the porphyrin ring. Although iron tetraphenylporphyrin complexes are frequently used as models for protoporphyrin- containing hemoproteins, one should be aware that such differences in the peripheral porphyrin substituents may significantly affect the spectral properties of the model complex.     

Supramolecular assemblies of a new class of nonplanar cationic metalloporphyrins and anionic metallophthalocyanines

The heteroaggregation behavior between a new class of nonplanar cationic β-octabrominated meso-alkylpyridinium zinc(II)-porphyrins (β-Br₈(ZnP)) and anionic tetrasulfonated metallophthalocyanines (MTSPc, M = Ni(II) and Cu(II)) has been studied by UV-Vis electronic spectroscopy, in dimethylsulfoxide (DMSO) solution. The heteroaggregate stoichiometry and the association constants were determined by means of Job plots. Dimers and unexpected trimers, taking into account the existence of axially coordinated DMSO molecules to the central metal in both β-Br₈(ZnP) and MTSPc complexes, are formed in solution. The spectroscopic properties of the heteroaggregates are markedly different from those observed in the correspondent planar cationic derivatives, the heteroaggregates showing major changes predominantly in the β-Br₈(ZnP) Soret band region and minor effects in the MTSPc Q bands. The observed changes in the Soret band region (red/blue shifts, decrease in the absorption intensities) depend on the nature of the alkyl substituent attached to the meso-pyridinium group. The greater versatility of the nonplanar porphyrins accommodating the meso-substituents in out-of-plane and in-plane conformations is proposed to explain the observed stoichiometries and the differences on the heteroaggregates spectroscopic properties for each β-Br₈ZnP compound. The likely conformations assumed by the meso-substituents in these β-Br₈(ZnP) compounds and its spectroscopic characteristics are in accordance with the participation of the substituents as the main factor on the extent of the observed red-shifted spectra in nonplanar porphyrins. The obtained association constants (K_IP) for the dimers and trimers are lower than those previously found for the similar planar cationic porphyrin systems, due to the lack of extensive π-π interactions and to the less effective approximation between the ionic groups, resulting in loosened heteroaggregates, particularly for the trimeric systems. Furthermore, the experimental results suggest that the NiTSPc is more distorted in DMSO solution than the CuTSPc derivative, favoring the interaction with the nonplanar β-Br₈(ZnP) compounds.     

Planorbis corneus haemoglobin. Circular dichroism and susceptibility to proteases.

The purified haemoglobin of Planorbis corneus was subjected to protease digestion and the resulting products characterised by gel filtration and detergent-gel electrophoresis. Small functional subunits of molecular weights approximately 20,000 were obtained corresponding to a single haem group, but multiples of this unit were also always obtained even at high proteolytic enzyme: haemoglobin ratios. This suggested that the subunits of the native molecule (one-tenth containing perhaps ten O2-binding sites) were made up of single-binding site domains linked by regions of polypeptide chains having different susceptibilities to proteases. The far ultraviolet CD of the native haemoglobin indicated the presence of a high helix content (75--80%) in the protein. The near ultraviolet and visible CD spectra of oxy- deoxy-, and CO-haemoglobin were reported. Planorbis haemoglobin CD was more like that of vertebrate haemoglobins than that of haemoblobins. Nevertheless the Soret CD of Planorbis oxyhaemoglobin had only about half the rotational strength of that of human haemoglobin A, and was halved again upon removal of the ligand. Also in contrast to Lumbricus and human haemoglobins there was only a small decrease in rotational strength in the 260 nm band when Planorbis oxyhaemoglobin was deoxygenated.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

Thalassemia and G-6-PD Deficiency in Chinese-Canadians: Admission Screening of a Hospital Population

Admission screening was performed on 684 Chinese-Canadian patients for thalassemia, abnormal hemoglobins and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. Thirty-six healthy Chinese adults were also studied. The incidence of beta-thalassemia minor (hemoglobin A₂ greater than 3.5%) was 3.8%. Presumptive alpha-thalassemia minor (demonstration of occasional red cells containing hemoglobin H inclusion bodies) was found in 6.7%. Two patients had findings consistent with alpha-beta-thalassemia. The incidence of G-6-PD deficiency (abnormal methemoglobin reduction test) in adult males was 4.7%. In a parallel study the incidence of hemoglobin Bart''s in 310 Chinese newborns was 6.8%. Two mutant hemoglobins were found — hemoglobin E and hemoglobin J (Bangkok).     

Stability of RNA hairpin loops: A 6 -C m -U 6

The thermodynamics and circular dichroism of a series of A₆-C_m-U₆ (m = 4, 5, 6 or 8) oligoribonucleotides have been studied. These molecules form intramolecular hairpin loops at low temperatures and therefore are useful models for similar structures which occur in larger, natural RNA molecules. The stability of the helix forming the stem of these loops was found to be considerably greater than an intermolecular helix with the same length and composition. The most stable loop is m = 6. The enthalpy for initiation of the loop is unfavorable; it ranges from + 24 kcal, for m = 4 to + 21 kcal, for m = 6. The maximum in stability for the C₆ loop and the large positive enthalpy for loop initiation are in disagreement with expectations from simple theories assuming a Gaussian distribution of end-to-end distances. Loop strain for m = 4 and m = 5 and the unstacking of the cytosines on loop formation are likely physical explanations for these thermodynamic data. The circular dichroism spectrum of cytosine residues in the C₆ and C₈ loops is very similar to the spectrum of single-stranded oligoribocytidylate. However, the cytosine residues in the C₅ loop have a very different circular dichroism spectrum from the corresponding oligo(C₅) spectrum. In accordance with the thermodynamic data, we conclude from the circular dichroism data that the C₅ loop has an altered conformation from the C₅ and C₈ loops.     

Heme binding by a bacterial repressor protein,the gene product of the ferric uptake regulation (fur) gene ofEscherichia coli

Thefur gene product, Fur, ofEscherichia coli is a repressor when it binds Fe(II). Since heme and iron metabolism are closely linked and Fur is rich in histidine, a ligand for heme, the binding of heme to Fur was investigated. The oxidized Fur-heme complex is stable and low spin with a Soret maximum at 404 nm and no 620-nm band. CO coordinates with the reduced heme-Fur complex, causing a shift from 412 nm to 410 nm, and stabilizes it, increasing the half-life from 5 to 15 min. Circular dichroism (CD) spectra in the Soret region show heme bound in an asymmetric environment in Fur, both in the oxidized and reduced-CO forms. Quenching of tyrosine fluorescence by heme revealed rapid, tight binding (K _d<1μM) with an unusual stoichiometry of 1 heme:1 Fur dimer. Fur binds Mn(II), a model ligand for the endogenous Fe(II), much more weakly (K _d>80μM). Far-ultraviolet CD spectroscopy showed that theα-helix content of apo-Fur decreases slightly with heme binding, but increases with Mn(II) binding. Competition experiments indicated that heme interacts with Fur dimers at the same site as Mn(II) and can displace the metal. In contrast to Mn(II), Zn(II) did not quench the tyrosine fluoroescence of Fur, affected the CD spectrum less than Mn(II), but did bind in a manner which prevented heme from binding. In sum, Fur not only binds heme and Zn(II) with sufficient affinity to be biologically relevant, but the interactions that occur between these ligands and their effects on Mn(II) binding need to be taken into account when addressing the biological function of Fur.     

Oxygen-binding characteristics of three extra-cellular haemoglobins of Artemia salina

The oxygen-binding characteristics of the three extracellular haemoglobins of brine shrimp (Artemia salina) were studied in vitro by using highly purified preparations. Haemoglobin I is induced last in the development of brine shrimps when functional gills are formed. It has the lowest oxygen affinity (p₅₀ 5.34mmHg), an intermediate Bohr effect (ø −0.09 at 20°C) above pH8 and a temperature-sensitivity (ΔH −44.8 to −45.6kJ/mol at pH8–9) comparable with those observed with other invertebrate haemoglobins [Weber & Heidemann (1977) Comp. Biochem. Physiol. A 57, 151–155]. Haemoglobin II, which is the first to be induced, soon after hatching of nauplius larvae, persists generally throughout the whole adult life. It has an intermediate oxygen affinity (p₅₀ 3.7mmHg), the highest Bohr effect (ø −0.21 at 20°C) above pH8 and a similar temperature-sensitivity (ΔH −46.0 to −54.8kJ/mol at pH8–9) as haemoglobin I. However, haemoglobin III, which is induced second several hours after the induction of haemoglobin II but disappearing from the haemolymph in the middle of adult life, has the highest oxygen affinity (p₅₀ 1.8mmHg), the lowest Bohr effect (ø −0.03 at 20°C) above pH8.5 and a high resistance against temperature variation between 10 and 25°C at pH8.5–9 (ΔH −22.6 to −23.0kJ/mol). At pH7.5–8, haemoglobin III exhibits a similar temperature-sensitivity under 30°C as do other haemoglobins. All three haemoglobins have a rather low co-operativity, with Hill coefficients (h 1.6–1.9 at pH8.5), which are dependent on both pH and temperature. The highest co-operativity was observed at 20°C and pH9 for haemoglobins I and II, whereas it was at 27°C and pH8.5 for haemoglobin III. Thus the oxygen-binding behaviour of haemoglobin III in vitro is significantly different from those of haemoglobins I and II and indicates possibly its specific physiological role in vivo in the adaptive process in the natural environment.     

The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

Coordination of Nitric Oxide by Heme—Hemopexin

Hemopexin, which acts as an antioxidant by binding heme (K _d < 1 pM), is synthesized by hepatic parenchymal cells, by neurons of the central and peripheral nervous systems, and by human retinal ganglia. Two key regulatory molecules, nitric oxide (·NO) and carbon monoxide (CO), both bind to heme proteins and since ferroheme–hemopexin binds CO, the possible role of heme–hemopexin in binding ·NO was investigated. ·NO binds rapidly to hemopexin-bound ferroheme as shown by characteristic changes in the Soret and visible-region absorbance spectra. Circular dichroism spectra of ·NO–ferroheme-hemopexin in the Soret region exhibit an unusual bisignate feature with a zero crossover at the absorbance wavelength maximum, showing that exciton coupling is occurring. Notably, the ·NO complex of ferroheme–hemopexin is sufficiently avid and stable to allow hemopexin to bind this molecule in vivo and, thus, hemopexin may protect against NO-mediated toxicity especially in conditions of trauma and hemolysis.     

Linear dichroism of electric field oriented bacteriochlorophyll a-protein from green photosynthetic bacteria

Bacteriochlorophyll a-protein from Prosthecochloris aestuarii strain 2K was oriented in a pulsed electric field. The room temperature linear dichroism spectrum of the oriented protein in the Q_y region of the bacteriochlorophyll a absorption exhibits a single asymmetrical peak at 813 nm with a shoulder extending to the blue. The ≈12 nm fullwidth of the linear dichroism peak is only about half that of the 300 K absorption spectrum. The linear dichroism at 813 nm was not saturated at field strengths of up to 15 kV/cm. The time dependence of the linear dichroism suggests that the orienting particles are aggregates of at least some tens of bacteriochlorophyll a-protein trimers. The linear dichroism peak coincides in wavelength with the 813-nm peak of the 300 K, 4th derivative absorption spectrum of the protein and is therefore attributed to the bacteriochlorophyll a Q_y exciton transition observed in absorption at the same wavelength.