Modification of isolated α and β chains of human hemoglobin by the metal tetrasulfonated phthalocyanines. Studies on hybrid phthalocyanine-heme intermediates

α and β chains of hemoglobin have been modified with cobalt(II) tetrasulfonated phthalocyanine in place of heme. They display properties very similar to those of iron(II) phthalocyanine modified α and β chains. Mixed together they form tetrameric cobalt(II) phthalocyanine hemoglobin.Incorporation of Co(II)L into α and β globins results in stabilization of the protein structure, which is shown by a marked increase in its helicity content. Cobalt phthalocyanine substituted α and β chains are able to combine reversibly with oxygen giving more stable oxygenated species than their native analogues. The rate of both processes is lower in the case of the modified α chain. Recombination of the phthalocyanine α and β chains with the alternate heme containing chains give tetrameric hybrid hemoglobins. These comprise two phthalocyanine modified subunits and two heme containing subunits. The helicity content of the tetrameric hybrid hemoglobin calculated for one subunit is lower that the arithmetic mean of helicities for its isolated subunits. This suggests a destabilizing chain-chain interaction within the tetramer. Unlike in the separated subunits, oxygen binding by hybrid hemoglobins is irreversible. Deoxygenation by argon bubbling leads to the formation of inactive species which in oxygen atmosphere undergo irreversible oxidation with destruction of the complex.     

The mitochondrial genome is large and variable in a family of plants (cucurbitaceae)

The genome sizes of mitochondrial DNA from darkgrown (etiolated) shoots of several higher plants were determined by reassociation kinetics and restriction analysis. Kinetic complexities obtained from reassociation kinetics measured spectrophotometrically indicate a mitochondrial genome size of 1600 Md for muskmelon, 1000 Md for cucumber, 560 Md for zucchini squash and 220 Md for watermelon (four species in the cucurbit family), as well as 240 Md for pea and 320 Md for corn. The kinetic curves also reveal the presence (except in corn) of sequences of a few magadaltons of complexity, reiterated about 10-50 times and representing 5%- 10% of the DNA in each mitochondrial genome. Molecular weight summation of fragments resulting from digestion with restriction endonucleases Sal I and Kpn I give genome size estimates similar to those obtained from reassociation kinetics, except for muskmelon and cucumber, for which the large number of fragments of similar size limits our estimate to at least 500 Md. The number of mitochondrial genomes per diploid cell is estimated to be about 110 to 140 for muskmelon, zucchini and watermelon. We consider the possible evolutionary mechanisms by which the mitochondrial genome has grown within the cucurbit family and the possible reasons for the existance of a seven to eight-fold range in mitochondrial genome size among such closely related species.     

Intrapopulational differences of genome structures in two structures of salmon-like fishes

The natural populations of salmon-like catadromous fishes usually include several percents of residual (dwarf) forms, which are 10-15 times smaller than normal forms. A comparative investigation of normal and residual forms in two species: Oncorhynchus nerka and Salvelinus malma (Salmoniformes order) was made by means of DNA molecular hybridization technique. The essential differences in reassociation kinetics was detected in DNA from normal and residual forms of both species. The genome sizes (kinetic complexity) of normal and residual forms are approximately the same. But some families of repetitive nucleotide sequences are represented by considerably different amount of copies. Intrapopulational differences of genome structures of normal and residual forms in both species seem to be more rough in respect to reassociation kinetic than corresponding interspecies differences between malma and nerka. Comparative analyses and reassociation kinetics for long and shrt DNA fragments imply "xenopus type" of nucleotide sequence organization in both species which is common for the majority of animal and plant genomes investigated.     

Structural characteristics (reassociation and melting kinetics) of kinetoplast DNA from Crithidia oncopelti

A highly purified associate of kinetoplast DNA is isolated from C. oncopelti, and its physico-chemical properties are studied. Both native associate and its ultrasonic fragments are found to have a complex character of melting. 5-6 melting zones (3 of them being the main) are found on the melting curve. Analysis of reassociation kinetics of sonicated associate of kinetoplast DNA has revealed the presence of at least two components: fast reassociating component (65-70% of complex DNA), which reassociation kinetics is equivalent to the unique sequence with molecular weight of 2.3. - 10(6) daltons, and slow reassotiating component (15% of complex DNA), having reassociation kinetics equivalent to unique sequence of 26 - 10(6) daltons. The data obtained suggest that complex associate of kinetoplast DNA is heterogenous for its nucleotide sequence and base composition.     

DNA reassociation kinetics in diploid and phylogenetically tetraploid cyprinidae.

Four diploid and three phylogenetically tetraploid Cyprinidae (Ostariophysi) have been characterized as for nuclear DNA content, modal chromosome number and DNA reassociation kinetics (hydroxyapatite chromatography). Among the diploid species nuclear DNA content (10(-12) g DNA/2C) was 1.62 for Tinca tinca, 1.87 for Scardinius erythrophthalmus, 2.53 for Leuciscus cephalus and 2.75 for Alburnus alburnus, while the phylogenetically tetraploid species Carassius auratus, Barbus barbus and Cyprinus carpio attained 3.40, 3.66 and 3.80 respectively. Modal chromosome number was 2n = 48-50 for diploid individuals and 2n = 100-104 for phylogenetically tetraploid ones. In all the species 5--8% of the genome is represented by highly repetitive and foldback DNA. In DNA reassociation kinetics of phylogenetically tetraploid Cyprinidae a distinct plateau separates an intermediate reassociating sequence fraction (about 22% of the genome; with average repetition frequencies between 1,000 and 1,400) from a slow reassociating one (unique DNA; about 72% of the genome). These two genome fractions are not clearly distinguishable from each other in Cot curves of the diploid Cyprinidae, where a similar plateau is not evident. Since simple ploidy changes are not expected to affect DNA reassociation kinetics we suggest a different evolution in the genome organization of the two ploidy groups. Some possible hypotheses are discussed.     

A weak Fe-O bond in the oxygenated complex of the nitric-oxide synthase of Staphylococcus aureus

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate L-arginine and the cofactor H(4)B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form (Fe(III)O(2)(-)) with a single nu(O-O) mode at 1135 cm(-1). In the presence of L-arginine, an additional nu(O-O) mode at 1123 cm(-1) was observed, indicating an increased pi back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the nu(Fe-O) mode of a NOS has been observed. The low frequency of this mode, at 517 cm(-1), points to an oxygenated complex that differs from that of P450(cam). The electronic structure of the oxygenated complex and the effect of L-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.     

Genome structure in higher plants. Reassociation kinetics of DNA from Vicia faba and Vicia sativa

The work been concerned with a study of the kinetics of reassociation of total DNA and that of the fraction of unique sequences in plants from the Vicia family, i. e. Vicia faba and Vicia sativa. The size of the genome was determined by the kinetics of reassociation of the DNA of the fraction of unique sequences and the amount of DNA per nucleus was determined cytophotometrically. It has been shown that the size of the genome expressed in C(0)t units and the size expressed in gramms are not the same which testifies to the absence of true unique genes in the genome of the species studied. The analysis of the possible methodical errors was carried out. On the basis of the data obtained a suggestion was made of a model of chromosomes organization including 12 units of polynemization for Vicia faba and 4 units for Vicia sativa.     

Reaction of S-nitrosoglutathione with the heme group of deoxyhemoglobin

The mechanism of interaction between S-nitrosoglutathione (GSNO) and hemoglobin is a crucial component of hypotheses concerning the role played by S-nitrosohemoglobin in vivo. We previously demonstrated (Patel, R. P., Hogg, N., Spencer, N. Y., Kalyanaraman, B., Matalon, S., and Darley-Usmar, V. M. (1999) J. Biol. Chem. 274, 15487-15492) that transnitrosation between oxygenated hemoglobin and GSNO is a slow, reversible process, and that the reaction between GSNO and deoxygenated hemoglobin (deoxyHb) did not conform to second order reversible kinetics. In this study we have reinvestigated this reaction and show that GSNO reacts with deoxyHb to form glutathione, nitric oxide, and ferric hemoglobin. Nitric oxide formed from this reaction is immediately autocaptured to form nitrosylated hemoglobin. GSNO reduction by deoxyHb is essentially irreversible. The kinetics of this reaction depended upon the conformation of the protein, with more rapid kinetics occurring in the high oxygen affinity state (i.e. modification of the Cysbeta-93) than in the low oxygen affinity state (i.e. treatment with inositol hexaphosphate). A more rapid reaction occurred when deoxymyoglobin was used, further supporting the observation that the kinetics of reduction are directly proportional to oxygen affinity. This observation provides a mechanism for how deoxygenation of hemoglobin/myoglobin could facilitate nitric oxide release from S-nitrosothiols and represents a potential physiological mechanism of S-nitrosothiol metabolism.     

Carbon dioxide and oxygen linkage in human hemoglobin tetramers

Differential binding curve measurements for oxygen in the presence of fixed carbon dioxide activities have allowed a detailed determination of the linkage between carbon dioxide and the oxygenated intermediates of human hemoglobin. Model-independent analysis of the data shows that at pH 7.4: (1) the oxygen binding curves are asymmetrical, the population of the triply oxygenated species being negligible; (2) the shape of the oxygen binding curve is invariant with carbon dioxide activity; (3) the maximum linkage is -0.32 moles carbon dioxide per mole oxygen; and (4) the overall carbon dioxide-dependent shift in the oxygen binding curve cannot be explained in terms of carbamino formation alone, the additional influence of bicarbonate being required. An allosteric model that accounts for the low population of triply oxygenated hemoglobin species is employed here as a framework from which to explore the carbon dioxide linkage mechanism at the intermediate stages of oxygenation. Carbon dioxide binding constants are found to be 780 M-1 and 580 M-1 for carbon dioxide binding to the deoxygenated alpha and beta chains, respectively, and 150 M-1 for carbon dioxide binding to the oxygenated form of both chains, as determined by simultaneous fitting of the oxygen binding curves with the model. Finally, by use of the determined binding polynomial for the carbon dioxide-oxygen linkage scheme, we have constructed a series of linkage graphs.     

Differential regulation of hexameric and dodecameric hemocyanin from A. leptodactylus

The oxygen binding properties of hemocyanins are regulated on a short time scale by effectors such as l-lactate, urate and protons, and on longer time scales by expression of the different types of subunits. For Astacus leptodactylus it was shown previously that acclimation to higher temperatures leads to increased levels of a 6-meric hemocyanin species, whereas at lower temperatures the 12-meric form prevails. Here we show that the temperature dependence of the two forms supports the idea, that the maintenance of high affinity towards oxygen is the driving force for the differential expression of these hemocyanins. Furthermore, the two different types of hemocyanin differ not only in the affinity to oxygen, but also with respect to their interaction with l-lactate: while the 12-meric form displays a normal shift in oxygen affinity upon the addition of l-lactate this allosteric regulation is absent in the 6-meric form. Exclusive binding of l-lactate to the 12-meric form was supported by isothermal titration calorimetry. These results indicate that l-lactate binds either at the interface between the two hexamers or at subunit α′ which is responsible for the formation of the 12-mers and is not present in the 6-meric form. Urate has a comparable effect on the oxygen affinity of 6-meric and 12-meric forms and also binds to a similar extent to the oxygenated state as determined by isothermal titration calorimetry. Thus, urate and l-lactate do not seem to share the same binding sites. Interestingly, urate binding sites with no allosteric effect seem to exist, which is unusual. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Ligand binding, reactivity and biological activity of a distal pocket mutant of neuroglobin

We have generated the Lys67Glu mutant form of neuroglobin. Experimental spectral studies are consistent with a six coordinate heme in which the distal histidine bond is stretched compared to the wild type protein. Carbon monoxide binding to the ferrous form of the mutant follows a hyperbolic concentration dependence limiting at the histidine dissociation rate of 0.7 s(-1). Further analysis indicates a significantly lowered histidine binding constant. Oxygen binding kinetic studies confirm the higher heme ligand dissociation level and indicate a p50 value for oxygen binding<1 mmHg. The ferrous form of the protein yields an oxygenated intermediate on reaction with oxygen. The rate of oxidation, by oxygen, follows a complex concentration dependence, consistent with the presence of two distinct oxidation mechanisms. A quantitative model for the two oxidation processes has been developed, which is consistent with a lowered distal histidine binding constant in the mutant form of the protein. These data suggest that the protein structure surrounding the heme site in neuroglobin limits access to external ligands and provides an energy barrier to the structural changes following ligand binding in this protein. However, the mutation does not appear to affect reactivity with cytochrome c and the anti-apoptotic activity of the mutant in human cells of neuronal origin is increased as compared to the wild type protein.     

Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD

Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The kinetics of the reconstitution reaction have been measured by 1) enzyme activity assays, 2) spectrophotometric assays to measure FAD binding, and 3) high performance liquid chromatography analysis measuring the distribution of monomeric, dimeric, and tetrameric species during reconstitution. The kinetic analysis indicates that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway.     

Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. The activity of Ras is up-regulated by cellular agents, including both protein (guanine nucleotide exchange factors) and redox-active agents (nitric oxide (NO) and superoxide anion radical (O2*). We have recently elucidated the mechanism by which NO promotes guanine nucleotide dissociation of redox-active NKCD motif-containing Ras and Ras-related GTPases. In this study, we show that guanine nucleotide dissociation is enhanced upon exposure of the redox-active GTPases, Ras and Rap1A, to O2* and provide evidence for the efficient guanine nucleotide reassociation in the presence of the radical quenching agent ascorbate to complete guanine nucleotide exchange. In vivo, guanine nucleotide reassociation is necessary to populate Ras in its biologically active GTP-bound form after the dissociation of GDP. We further show that treatment of the redox-active GTPases with O2* releases GDP in form of an unstable the oxygenated GDP adduct, putatively assigned as 5-oxo-GDP. 5-Oxo-GDP was not produced from either the C118S or the F28L Ras variants upon the treatment of O2*, supporting the involvement of residues Cys118 and Phe28 in O2*-mediated Ras guanine nucleotide dissociation. These results indicate that the mechanism of O2*-mediated Ras guanine nucleotide dissociation is similar to that of NO/O2-mediated Ras guanine nucleotide dissociation.     

Effects of Sequence Divergence on the Reassociation Properties of Repetitive DNAs

PYRIMIDINE tract analysis of two satellite DNAs suggests that their basic sequences are simpler than those calculated from their rates of reassociation (réf. 1 and unpublished results of A. Carr-Brown, E. M. S. and P. M. B. Walker). One possible explanation for this discrepancy would be that mismatched base pairs, which are known to affect the stability of duplexes², might have a serious effect on the rate of reassociation of DNA. The work of Sutton and McCallum³ (see following article³) shows that this is indeed the case for mouse satellite DNA. If this effect were general it would have serious consequences for the interpretation of the reassociation kinetics of the repeated sequences in higherorganism DNA, because the bulk of these sequences reassociate to give badly matched duplexes. I now discuss a simple modification of the mechanism for DNA reassociation put forward by Wetmur and Davidson⁵, which may explain why the effect of mismatching is so large and which suggests a relationship which may eventually be used to correct for the effect.     

Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigated. Sedimentation equilibrium data indicate that the ferrous deoxygenated and carbonylated derivatives display low values of equilibrium dimerization constants, 6 x 10(2) and 1 x 10(2) M(-1), respectively, at pH 7.0 and 10 degrees C. The behavior of the oxygenated species, as measured in sedimentation velocity experiments, is superimposable to that of the carbonylated derivative. The kinetics of O(2) combination, measured by laser photolysis at pH 7.0 and 20 degrees C, is characterized by a second-order rate constant of 2 x 10(8) M(-1) s(-1) whereas the kinetics of O(2) release at pH 7.0 is biphasic between 10 and 40 degrees C, becoming essentially monophasic below 10 degrees C. Values of the first-order rate constants (at 20 degrees C) and of the activation energies for the fast and slow phases of the Vitreoscilla Hb deoxygenation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 kcal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb deoxygenation is unrelated to the association state of the protein. The observed biphasic oxygen release may be accounted for by the presence of two different conformers in thermal equilibrium within the monomer. The two conformers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB10 and a structure in which such interaction is absent. The slow interconversion between the two conformers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices C and E.     

An oxygen-binding flavohemoprotein from Alcaligenes eutrophus.

A procedure is described for the purification of a soluble flavohemoprotein from the hydrogen bacterium Alcaligenes eutrophus. The isolated protein exists as a monomer with a molecular weight of approx. 43,000. The molecule contains two prosthetic groups, 1 mol each of noncovalently bound FAD and protoheme per monomer. The absorption spectra of the protein in its ferric, ferrous-deoxy and ferrous-carboxy forms are similar to those of hemoglobins, with the exception of the flavin contribution (absorption maxima--ferric form: 395, 456, 483, 645 nm; ferrous-deoxy form: 436, 560 nm; ferrour-CO form: 423, 539, 569 nm). The flavohemoprotein when reduced by NADH in aerobic solution is capable of binding oxygen reversibly. The stable oxygenated complex exhibits absorption maxima at 414, 541, and 576 nm. The protein catalyzes the reduction of various dyes and cytochrome c by NADH.     

Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.     

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.     

Carboxylic acids from hairpencils of male Amauris butterflies (Lep.: Danainae)

63 Carboxylic acids were identified from the male hairpencils of four species of the genus Amauris (Lep.: Danainae), namely A. echeria (Stoll), A. hecate (Butler), A. ochlea (Boisduval) and A. albimaculata Butler. Straight chain saturated as well as unsaturated carboxylic acids, some of which containing an additional oxygen function, contribute to the species-specificity of the odour bouquets. Oxygenated fatty acids form a new class of insect volatiles, 5 of the 10 ketoacids found represent new natural products. (E)-7-Oxo-11-tetradecenoic acid is the main volatile component of the hairpencils of A. echeria, the species with the highest amount of oxygenated fatty acids (70% of the extractable volatiles). 9-Hydroxyoctadecanoic acid is a major compound in both A. ochlea and A. albimaculata while in A. hecate oxygenated carboxylic acids are present in minute amounts only.     

Use of DNA reassociation in bacterial classification

The reassociation properties of DNA provide invaluable taxonomic tools. Different methods may give different reassociation values. However, the thermal stability of reassociated DNA strands (a measurement that seems independent of method) is useful in delineating genomic species. Although many phenotypically defined species have been confirmed by DNA reassociation, some medically important genomic species previously had been split into several nomenspecies on the basis of a few characteristics whereas some environmental genomic species had been lumped into unidentifiable aggregates. It might take some time before the nomenclature can be adapted to new taxonomic findings.