Functional multiplicity and structural correlations in the hemoglobin system of larvae of Chironomus thummi thummi (Insecta, Diptera): Hb components CTT I, CTT II beta, CTT III, CTT IV, CTT VI, CTT VIIB, CTT IX and CTT X

Larvae of the dipteran insect Chironomus thummi thummi that burrow in fresh-water muds, contain at least 12 hemoglobin (Hb) components of which the functional properties have not been systematically documented, although their amino acid sequences have been elucidated, showing mutually distinct primary structures. We isolated eight components (the monomeric Hbs CTT I, CTT III and CTT IV and the dimeric Hbs CTT II beta, CTT VI, CTT VIIB, CTT IX and CTT X) and measured in each O2 affinity and cooperativity and their pH dependence, and the effects of temperature, NaCl and ATP. The O2 affinities, Bohr- and temperature effects of the isohemoglobins are discussed in relation to mode of life and the microenvironmental conditions to which the larvae are subjected in nature, and with regard to the molecular mechanisms underlying the Hb-oxygenation reactions.     

Hemoglobin in Chironomus ramosus (Insecta, Diptera): an electrophoretic study of polymorphism, developmental sequence and interspecific relationship

The larvae of the dipteran insect, Chironomus ramosus, found in Shillong, India, contain eleven (11) hemoglobin (Hb) components of which three are monomers (CI, CIV and CVI) and seven are dimers (CIII, CV, CVII, CVIII, CIX, CX and CXI), while one (CII) exists in both monomeric and dimeric states.Four monomeric components were isolated, purified and partially characterized. The N-terminal amino acids were determined and showed glycine for CI and leucine for the other components (CII, CIV and CVI).Three hemoglobin components were found to be present in all stages of larval development, except the first instar larvae. Some Hb components were synthesized in a particular instar, as revealed by electrophoretic appearance.Electrophoretic mobilities of seven components and N-terminal amino acid residues of two components of Hb were similar in both Chironomus ramosus and Chironomus thummi thummi.     

Allosteric effect of protons and adenosine triphosphate on hemoglobins from aquatic amphibia

Summary The analysis of hemoglobins from two anurans, one semi-aquatic (Leptodactylus labyrinthicus) and the other aquatic (Pipa carvalhoi), showed several components isolated by CM-cellulose chromatography. The two major components (Hb II and Hb III) ofL. labyrinthicus and the major components (Hb III and Hb IV) ofP. carvalhoi possess functional properties as follows: i.P. carvalnoi Hb III and Hb IV andL. labyrinthicus Hb II had very small Bohr effects (–0.05) and a substantial heterotropic effect with polyphosphates. ii.L. labyrinthicus Hb III produced a normal Bohr effect of –0.17, with no influence of polyphosphates.The authors wish to dedicate this paperin memoriam of Prof. Eraldo Antonini who contributed much to the knowledge of hemoglobins     

A nuclear overhauser study of heme orientational isomerism in monomeric Chironomus hemoglobins

The nuclear Overhauser effect (NOE) was used to investigate heme orientation and to obtain assignments for all resolved resonances in the 1H-NMR spectrum of met-cyano Chironomus thummi thummi monomeric hemoglobins III and IV (Hb III and Hb IV). The only non-heme resolved resonance was found to be from Phe-38 (CD1), and NOE dipolar connectivity between this resonance and the heme 5- and 8-methyls was used to establish the absolute orientation of the heme for each heme-insertion isomer present. The assignments of resonances and heme disorder permitted structural comparisons between the various components, including those due to a point mutation in Hb III. Finally, the characteristic differences of NOE patterns to amino-acid protons from substituents on heme pyrroles I and II formed the basis for assigning resonances and heme orientation relative to native Hb IV for deuterohemin-reconstituted Hb IV, for which there are no X-ray data available.     

Hemoglobins,XLVIIII

Summary Hagfish hemoglobin has three main components, one of which is Hb III. It is monomeric and consists of 148 amino acid residues (M = 17 350). Its complete primary structure, previously published, is discussed here. The proximal amino acid (F8) of the heme linkage is histidine as always in the hemoglobins, but the regularly expected distal histidine E7 is substituted by glutamine. This substitution, leading to a new kind of heme linkage, has hitherto only been demonstrated in opossum hemoglobin. It is suggested that E7, Gln, is directed out of the heme pocket, and that the adjacent Ell, Ile, is directed toward the inside of the pocket, giving the distal heme contact instead of histidine.Myxine Hb III has an additional tail of 9 amino acid residues at its N-terminal end, as has the hemoglobin ofLampetra fluviatilis. The genetic codes ofMyxine andLampetra hemoglobins show 117 differences, in spite of many morphological resemblances between hagfish and lamprey. Their primary hemoglobin structures show differences substantial enough to bo compatible with the divergence of the two families some 400–500 million years ago.     

Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions

The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.     

Iron-histidine stretching vibration in the deoxy state of insect hemoglobins with different O2 affinities and Bohr effects

Resonance Raman spectroscopy has been employed to detect the iron-proximal histidine stretching mode in deoxyhemoglobins from insect larvae of Chironomus thummi thummi (CTT). With the excitation of 413.1 nm, we observe a sharp and intense line in the 220-224 cm-1 region. The assignment of this line to the Fe-N epsilon (His) stretching mode was made on the basis of a 3-cm-1 shift upon 57Fe/54Fe isotope substitution. The Fe-N epsilon (His) vibration is used to monitor the possible changes in the Fe-N epsilon (His) bond strength (hence bone length) in the deoxy state of the monomeric (CTT I, III, and IV) and dimeric (CTT II) insect hemoglobins. As these hemoglobins differ in O2 affinity, off-rate and on-rate constants, and in the Bohr effect, they are excellent model systems for investigating the mechanism of protein control of the heme reactivity. Some of these hemoglobins (CTT III, IV, and II) are allosteric, exhibiting two interconvertible conformational states with high and low O2 affinity at high and low pH, respectively. The Fe-N epsilon (His) stretching frequency does not correlate with the O2 affinity, the on-rate and the off-rate constants for different hemoglobins, for different conformational states, and for modified hemoglobins with different heme peripheral groups. This vibrational mode is insensitive to deuteration of the heme vinyl groups. It is important to note that the Fe-N epsilon (His) bonds in the high pH (high-affinity) and the low pH (low-affinity) states are identical. This implies that the O2 molecule, prior to binding, "sees" identical binding sites. Thus, the difference in free energy changes upon O2 binding is manifested only in the oxy form.     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Comparison of insect hemoglobins (Erythrocruorins) from Chironomus thummi thummi and Chironomus thummi piger (Diptera). The primary structure of the monomeric hemoglobin CTP III

The monomeric hemoglobin fractions of Chironomus thummi thummi (CTT) and Chironomus thummi piger (CTP) differ in the ratio of their components. The determination of the primary structure of the component CTP III was achieved by automatic Edman degradation of the native chain, the tryptic peptides and the C-terminal fragment, obtained by cleavage at the single tryptophan residue. It revealed two chains in the ratio 1:1 which share the ambiguity threonine/isoleucine in position 57 with CTT III. Whereas one chain is identical to the CTT III hemoglobin, the other differs in having isoleucine in position 105 and alanine in position 134. The CTP monomeric hemoglobin fraction comprises 8% of a component (CTP IV A) with a more negative charge than CTT IV but with an identical sequence up to position 44. This study reveals a very high polymorphism within Chironomus species and points out the need for more data at the gene level in order to provide better understanding of this striking phenomenon.     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Ligand-dependent Bohr effect of Chrionomus hemoglobins.

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation. For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2-liganded and CO-liganded states of hemoglobin III. Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi; here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively. On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine-E11, obstructing the movement of the heme-iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved. The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.     

The hemoglobins of phoronopsis viridis, of the primitive invertebrate phylum phoronida: characterization and subunit structure.

The primitive invertebrate, Phoronopsis viridis, of the phylum Phoronida, has intra-cellular hemoglobins composed of four unique polypeptide chains, two of which associate to form hetero- and homodimers and two which do not associate at all. The CO-derivatives of the associating chains are completely dimeric; removal of the ligand does not result in further aggregation as it does in several other invertebrate hemoglobins. Oxidation of the associating hemoglobins is accompanied by dissociation to monomers, but the cyanide derivative of the methemoglobin is dimeric. The four polypeptide chains all have molecular weights of about 16,000 as determined by iron content and gel electrophoresis with sodium dodecyl sulfate. The two associating chains form three components with isoelectric points at pH 5.6, 5.9, and 6.9 whereas those for the two monomeric chains are at pH 6.2 and 7.9. The chains have been characterized by amino acid composition, tryptic peptide patterns, and the amino acid sequence of the NH₂-terminal segment. The oxygen equilibrium of a dimeric fraction has been determined at pH 7.5 and 20 °C; the pressure of half-saturation is 2.3 mm Hg.     

The primary structure of a dimeric hemoglobin (erythrocruorin); component CTT VI of Chironomus thummi thummi, Diptera (author's transl)]

The complete amino acid sequence of 147 residues was determined automatically for a major dimeric component (CTT VI) of the insect larva Chironomus thummi thummi (Diptera). The molweight was found to be 32411. All tryptic, maleylated tryptic and cyanogen bromide peptides were isolated. The handling of some large fragments was facilitated by maleylation and subsequent ion exchange chromatography. Some details of the primary structure are discussed. The alignment of the amino acid sequence with that of human alpha-chains shows only 29 identical positions.     

In situ localization of two haemoglobin gene clusters in the chromosomes of 13 species of Chironomus

Two haemoglobin (Hb) gene clusters cloned from the genomic DNA of Chironomus thummi were localized in the polytene chromosomes of 13 Chironomus species. The haemoglobin gene cluster containing the genes for the monomeric haemoglobin proteins III and IV (CttG1) hybridized in all species to the end of chromosome arm E. The haemoglobin gene cluster containing the genes for the dimeric HbVIIB proteins (piHb1) could be localized to chromosome arm D. The chromosomal position of the haemoglobin genes was always found in morphologically specified groups of bands and is in agreement with cytological data, which have been used to establish the evolutionary relationships between the species of the genus Chironomus.     

The cobalt-nitrosyl stretching vibration as a sensitive resonance Raman probe for distal histidine-nitrosyl interaction in monomeric hemoglobins

The Co-NO stretching vibration has been assigned in the resonance Raman spectra of various cobalt-substituted monomeric hemoglobins by employing isotope-labeling of nitrosyl (14N16O, 15N16O, 14N18O). Monomeric hemoglobins with a distal histidine (sperm whale myoglobin and leghemoglobin) exhibit this vibration at 573-575 cm-1, whereas hemoglobins without distal histidine (elephant myoglobin and insect hemoglobin from Chironomus thummi thummi, CTT III) show this vibration in the range of 553-558 cm-1. The Fe-NO stretching vibration which occurs in the range of 554-556 cm-1 does not reflect the distal histidine-ligand interaction. Therefore, the Co-NO moiety which is isoelectronic with the Fe-O2 moiety is a good monitor for distal effects on the exogenous ligand of hemoglobins, especially due to the fact that in hemoglobins with distal histidine the Fe-O2 stretching vibration (567-572 cm-1) is similar to the Co-NO stretching vibration.     

Amino acid sequence of alpha-chain of hemoglobin IV from trout (Salmo irideus)

The amino acid sequence of the alpha-chain of trout hemoglobin (Hb) IV is given, thus completing the primary structure of the hemoglobin component of trout's blood characterized by the Root effect. The trout Hb IV alpha-chain consists of 142 amino acid residues; comparison with the corresponding sequences from human and carp hemoglobins shows differences of 50.0 and 35.9%, respectively. A difference of 39.6% is found with the alpha-chain of trout Hb I, the other major hemoglobin component of trout blood, devoid of heterotropic effects.     

Bohr effect in monomeric insect haemoglobins controlled by O2 off-rate and modulated by haem-rotational disorder

The monomeric insect (Chironomus thummi thummi) haemoglobins CTT III and CTT IV show an alkaline Bohr effect. The amplitude of the Bohr effect curve of CTT IV is about twice as large as that of CTT III. In particular, at low pH a time-dependent 'slow' decrease in p50 upon cyclic oxygenation/deoxygenation is observed which is larger if dithionite, instead of ascorbate, is the reducing agent. The decrease of p50 (increase in affinity) correlates with the ratio of haem-rotational components exhibiting an increase of the 'myoglobin-like' haem-rotational component with high O2 affinity and high stability of the globin-haem complex. The replacement of protohaem IX by mesohaem IX and deuterohaem IX, respectively, causes an increase in O2 affinity following the order: proto less than meso less than deutero CTT Hbs. The Bohr effect, however, seems not to be affected by these porphyrin side-group substitutions. The O2 affinity is modulated by steric effects due to the substituents in position 2 and 4 via variation of the protein-haem interactions which influence the O2 release. The replacement of iron by cobalt in proto and meso CTT IV leads to an increase of the p50 by two to three orders of magnitude. Neither central metal nor vinyl replacement affect the Bohr effect. The natural CTT Hbs III and IV analyzed for mono-componential kinetic systems exhibit pH-dependent O2 off-rate constants: 300 s-1 (at pH 5.6) and 125 s-1 (at pH 9.7) for CTT III, and 550 s-1 (at pH 5.4) and 100 s-1 (at pH 9.0) for CTT IV. Inflection points and amplitudes of the log koff/pH plots correspond to those obtained from the Bohr effect curves indicating again a larger Bohr effect for CTT IV than for CTT III. In contrast, the O2 on-rate constants are pH-independent (kon = 1.15-1.26 X 10(8) M-1 s-1). Thus, the Bohr effect is completely controlled by the off-rate constants. Analysis for bi-componential kinetic systems employing the eigenfunction expansion method clearly identifies two kinetic components for proto-IX and deutero-IX CTT Hbs which can be attributed to the two haem-rotational components x and y (x and y differ due to an 180 degree rotation of the haem group about the alpha,gamma-meso axis; y is the myoglobin-like haem-rotational component).(ABSTRACT TRUNCATED AT 400 WORDS)     

Hemoglobin transition in relation to metamorphosis in normal and isogenicXenopus

Summary Electrophoretic separation of hemoglobins of normalXenopus laevis and of isogenic animals derived from female hybrids ofXenopus laevis×Xenopus gilli revealed 5–9 components in premetamorphic larvae, and 3–4 components in adult toads. InXenopus laevis the number of larval hemoglobin components showed considerable variation, but this variation was absent in isogenic tadpoles, suggesting a genetic basis for hemoglobin polymorphism in larvae.Electrophoretic separation of larval and adult hemoglobins at different concentrations of acrylamide and treatment of these solutions with mercaptoethanol revealed that larval hemoglobin components are charge isomers, whereas adult hemoglobin was found to contain a minor dimeric component.Estimation of hemoglobin components showed that the main increase in adult hemoglobin, i.e from 30–90% of total hemoglobin, occurs within 4 weeks after completion of metamorphosis. By incroporation of³H amino acids in vivo a switch to preferential synthesis of adult hemoglobin and a corresponding decrease in larval hemoglobin production could be demonstrated during early climax stages. This suggests that thyroid hormones are involved in the hemoglobin transition. Yet chemical inhibition of the larval thyroid by thiourea resulted in a delayed but complete hemoglobin transition without morphological transformation. It is concluded that hemoglobin transition and morphological transformation of theXenopus tadpole require different concentrations of thyroid hormones.Abbreviations Hb hemoglobin - Hb_A adult hemoglobin - Hb_L larval hemoglobin     

Extraordinary Conservation of Cysteines Among Homologous Chironomus Silk Proteins sp185 and sp220

Aquatic larvae of the midge, Chironomus tentans, synthesize a 185-kDa silk protein (sp185) with the cysteine-containing motif Cys-X-Cys-X-Cys (where X is any residue) every 20–28 residues. We report here the cloning and full-length sequence of cDNAs encoding homologous silk proteins from Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220). Deduced amino acid sequences reveal proteins of nearly identical mass composed of 72 blocks of 20–28 residues, 61% of which can be described by the motif X_5–8-Cys-X₅-(Trp/Phe/Tyr)-X₄-Cys-X-Cys-X-Cys. Spatial arrangement of these residues is preserved more than surrounding sequences. cDNA clones enabled us to map the genes on polytene chromosomes and identify for the first time the homolog of the Camptochironomus Balbiani ring 3 locus in Chironomus thummi. The apparent molecular weight difference between these proteins (185 vs 220 kDa) is not attributable to primary structure and may be due to differential N-linked glycosylation. DNA distances and codon substitutions indicate that the C. tentans and C. pallidivittatus genes are more related to each other than either is to C. thummi; however, substitution rates for the 5′- and 3′-halves of these genes are different. Blockwise sequence comparisons suggest intragenic variation in that some regions evolved slower or faster than the mean and may have been subjected to different selective pressures. Received: 30 August 1996 / Accepted: 6 November 1996