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PubMed1995

Slower aging in women: a proposed evolutionary explanation.

Differential female longevity is so far unexplained in evolutionary terms. The theory of evolutionarily necessary aging which goes back to Wallace appears to be up to the task. In this theory, aging minimizes competition between forebear and offspring. The aging equation which is implicit contains the well-known empirical Gompertz law as a special case. Moreover, its parameters are automatically sex-specific. It is shown that the slower aging of the female members of two species of mammals, humans and sperm whales, can be 'predicted' on the basis of this equation. A prediction of effective human monogamy under archaic conditions is obtained as a corollary. The analogous if opposite prediction for sperm whales (strong promiscuity) is empirically testable.

Aging↗
PubMed2006

Identification of highly brominated analogues of Q1 in marine mammals.

Three novel halogenated organic compounds (HOCs) have been identified in the blubber of marine mammals from coastal New England with the molecular formulae C(9)H(3)N(2)Br(6)Cl, C(9)H(3)N(2)Br(7), and C(9)H(4)N(2)Br(5)Cl. They were identified using high and low resolution gas chromatography mass spectrometry (GCMS) and appear to be highly brominated analogues of Q1, a heptachlorinated HOC suspected to be naturally produced. These compounds were found in Atlantic white sided dolphin (Lagenorhynchus acutus), bottlenose dolphin (Tursiops truncatus), common dolphin (Delphinus delphis), Risso's dolphin (Grampus griseus), harbor porpoise (Phocoena phocoena), beluga whale (Delphinapterus leucas), fin whale (Balaenoptera physalus), grey seal (Halichoerus grypus), harp seal (Phoca groenlandica) and a potential food source (Loligo pealei) with concentrations as high as 2.7 microg/g (lipid weight). The regiospecificity of C(9)H(3)N(2)Br(6)Cl is suggestive of a biogenic origin. Debromination of C(9)H(3)N(2)Br(6)Cl may be significant in the formation of C(9)H(4)N(2)Br(5)Cl.

Animals↗
PubMed1987

Isocyanide binding kinetics to monomeric hemoproteins. A study on the ligand partition between solvent and heme pocket.

The kinetics of methyl-, ethyl-, iso-propyl-, and ter-butyl-isocyanide binding to Aplysia limacina myoglobin (distal His----Lys) and the isolated beta chains from hemoglobin Zurich (distal His----Arg) have been investigated by flash photolysis at various temperatures above 0 degrees C. Sperm whale (Physter catodon) myoglobin and the isolated beta chains from normal adult hemoglobin have been used as references. In most reaction systems investigated the apparent extent of photolysis increases with temperature. For sperm whale myoglobin and the normal beta chains the increase is of the same magnitude and not correlated to the type of ligand used. On the contrary, for the two proteins lacking the distal histidine, the phenomenon is dependent on the size of the alkyl side chain of the ligand. The results, analyzed on the basis of the multibarrier model (Austin, R.H., K.W. Beeson, L. Eisenstein, H. Frauenfelder, and I.C. Gunsalus, 1975, Biochemistry, 16:5355-5373), suggest that the partition of the ligand molecules between the solvent and the heme pocket, occurring during the photolysis process, is primarily determined by interactions between the ligand and residues in the heme cavity rather than by diffusion through the protein matrix.

Animals↗
PubMed1996

Structural determinants of fluoride and formate binding to hemoglobin and myoglobin: crystallographic and 1H-NMR relaxometric study.

The x-ray crystal structure of the fluoride derivative of ferric sperm whale (Physeter catodon) myoglobin (Mb) has been determined at 2.5 A resolution (R = 0.187) by difference Fourier techniques. The fluoride anion, sitting in the central part of the heme distal site and coordinated to the heme iron, is hydrogen bonded to the distal His(64)E7 NE2 atom and to the W195 solvent water molecule. This water molecule also significantly interacts with the same HisE7 residue, which stabilizes the coordinated fluoride ion. Moreover, fluoride and formate binding to ferric Aplysia limacina Mb, sperm whale (Physeter catodon) Mb, horse (Caballus caballus) Mb, loggerhead sea turtle (Caretta caretta) Mb, and human hemoglobin has been investigated by 1H-NMR relaxometry. A strong solvent proton relaxation enhancement is observed for the fluoride derivatives of hemoproteins containing HisE7. Conversely, only a small outer-sphere contribution to the solvent relaxation rate has been observed for all of the formate derivatives considered and for the A. limacina Mb:fluoride derivative, where HisE7 is replaced by Val.

Animals↗
PubMed1997

The tautomeric state of histidines in myoglobin.

1H-15N HMQC spectra were collected on 15N-labeled sperm whale myoglobin (Mb) to determine the tautomeric state of its histidines in the neutral form. By analyzing metaquoMb and metcyanoMb data sets collected at various pH values, cross-peaks were assigned to the imidazole rings and their patterns interpreted. Of the nine histidines not interacting with the heme in sperm whale myoglobin, it was found that seven (His-12, His-48, His-81, His-82, His-113, His-116, and His-119) are predominantly in the N epsilon2H form with varying degrees of contribution from the Ndelta1 H form. The eighth, His-24, is in the Ndelta1H state as expected from the solid state structure. 13C correlation spectra were collected to probe the state of the ninth residue (His-36). Tentative interpretation of the data through comparison with horse Mb suggested that this ring is predominantly in the Ndelta1H state. In addition, signals were observed from the histidines associated with the heme (His-64, His-93, and His-97) in the 1H-15N HMQC spectra of the metcyano form. In several cases, the tautomeric state of the imidazole ring could not be derived from inspection of the solid state structure. It was noted that hydrogen bonding of the ring was not unambiguously reflected in the nitrogen chemical shift. With the experimentally determined tautomeric state composition in solution, it will be possible to broaden the scope of other studies focused on the electrostatic contribution of histidines to the thermodynamic properties of myoglobin.

Animals↗
PubMed2001

Genetic divergence and reproductive isolation between Anisakis brevispiculata and Anisakis physeteris (Nematoda: Anisakidae)s.

In order to assess the taxonomic status of Anisakis brevispiculata Dollfus, 1966 population samples of this taxon from central and south-eastern Atlantic ocean were compared at 22 enzymatic loci with samples belonging to Anisakis physeteris Baylis, 1923 from the Mediterranean sea and central-eastern Atlantic ocean. Very low interpopulational genetic divergence was observed both within A. brevispiculata (average D(Nei) = 0.008) and within A. physeteris (D(Nei) = 0.009) despite the geographic distance among the samples, indicating high levels of gene flow in both taxa. On the other hand, the average genetic distance between A. brevispiculata and A. physeteris was found to be D(Nei) = 0.80, a value generally observed between well differentiated congeneric species. The reproductive isolation between A. brevispiculata and A. physeteris is indicated by the following observations: (1) no F(1) hybrids or recombinant genotypes were until now observed; and (2) the two Anisakis species do not seem to share their definitive hosts. The main definitive host of A. brevispiculata is the pygmy sperm whale (Kogia breviceps), while for A. physeteris it is the sperm whale (Physeter catodon). Only adult males differ slightly in spicule length, while females and larval stages are not differentiated morphologically. Both A. brevispiculata and A.physeteris show a type II larva. The correct recognition of A. brevispiculata from A. physeteris and from other Anisakis species studied, in either sexes and at any life stage, is made easy by allozyme markers (e.g. Icdh, Gapdh, Sod-1, Np, Aat-2, Adk-2, fEst-2, PepB, PepC-2, Mpi). Diagnostic keys, which can be used for routine identification in the field of these Anisakis worms, based on genetic markers, are given.

Animals↗
PubMed1975

Scanning isoelectric focusing in small density-gradient columns. IV. The use of deuterium oxide for preparing the density gradient and its effects on isoelectric points of proteins.

The use of deuterium oxide as a substitute for sucrose in preparing density gradients for small isoelectrofocusing columns has been investigated. A density gradient was created directly in a 1.5-ml column by free inter-diffusion of three deuterium oxide solutions for 3 min. The resulting deuterium oxide concentration course (as shown by measurement of the refractive-index gradient) had a very high degree of linearity. Test runs with beta-lactoglobulin and sperm-whale myoglobin showed that the strength and stability of the deuterium oxide density gradient should normally be sufficient for stabilization of protein zones against convection during isoelectric focusing. The isoelectric points of beta-lactoglobulins A and B were found to increase as the concentration ratio of deuterium oxide to water at focusing level increased. Within the limits of experimental error, the pI shift corresponding to a given change in solvent composition was equal for both components. These findings are accounted for in terms of the deuterium isotope effect on the dissociation constants of protolytic groups and the shift in the asymmetry potential of the glass electrode in deuterium oxide as compared with water. The spontaneous reduction of sperm-whale metmyoglobin to ferrous myoglobin, which occurs on prolonged isoelectric focusing, is discussed, as are the benefits and drawbacks of deuterium oxide as a density-gradient solute.

Animals↗
PubMed2002

Enantiomer fractions of chiral organochlorine pesticides and polychlorinated biphenyls in standard and certified reference materials.

Enantiomeric ratios (ERs) and enantiomeric fractions (EFs) of a number of chiral organochlorine pesticides and PCB atropisomers were measured by chiral gas chromatography/mass spectrometry (GC/MS) in five standard (SRM) and certified (CRM) reference materials: SRM 1588a (organics in cod liver oil), SRM 1945 (organics in whale blubber), Marine Mammal Quality Assurance Exercise Control Material IV (NIST IV, organics in whale blubber), CRM trout, and CRM EC-5 (sediment). Target analytes were cis- and trans-chlordane, heptachlor exo-epoxide, oxychlordane, U82, MC5, MC6, MC7, o,p'-DDT, and PCB congeners 91, 95, 136, 149, 174, 176, and 183. Measured ERs and EFs are in close agreement with the few literature values reported for some of these analytes in SRMs and CRMs. Chiral PCB ERs and EFs measured by one-dimensional chiral GC/MS were similar to values measured using multidimensional chiral GC/MS. Non-racemic chiral compositions are in agreement with known uptake and biotransformation in the respective environmental matrices. These values should aid in the quality assurance/quality control methodologies for chiral environmental chemistry using standardized reference materials.

Adipose Tissue↗
PubMed2002

Ecology of southern ocean pack ice.

Around Antarctica the annual five-fold growth and decay of sea ice is the most prominent physical process and has a profound impact on marine life there. In winter the pack ice canopy extends to cover almost 20 million square kilometres--some 8% of the southern hemisphere and an area larger than the Antarctic continent itself (13.2 million square kilometres)--and is one of the largest, most dynamic ecosystems on earth. Biological activity is associated with all physical components of the sea-ice system: the sea-ice surface; the internal sea-ice matrix and brine channel system; the underside of sea ice and the waters in the vicinity of sea ice that are modified by the presence of sea ice. Microbial and microalgal communities proliferate on and within sea ice and are grazed by a wide range of proto- and macrozooplankton that inhabit the sea ice in large concentrations. Grazing organisms also exploit biogenic material released from the sea ice at ice break-up or melt. Although rates of primary production in the underlying water column are often low because of shading by sea-ice cover, sea ice itself forms a substratum that provides standing stocks of bacteria, algae and grazers significantly higher than those in ice-free areas. Decay of sea ice in summer releases particulate and dissolved organic matter to the water column, playing a major role in biogeochemical cycling as well as seeding water column phytoplankton blooms. Numerous zooplankton species graze sea-ice algae, benefiting additionally because the overlying sea-ice ceiling provides a refuge from surface predators. Sea ice is an important nursery habitat for Antarctic krill, the pivotal species in the Southern Ocean marine ecosystem. Some deep-water fish migrate to shallow depths beneath sea ice to exploit the elevated concentrations of some zooplankton there. The increased secondary production associated with pack ice and the sea-ice edge is exploited by many higher predators, with seals, seabirds and whales aggregating there. As a result, much of the Southern Ocean pelagic whaling was concentrated at the edge of the marginal ice zone. The extent and duration of sea ice fluctuate periodically under the influence of global climatic phenomena including the El Niño Southern Oscillation. Life cycles of some associated species may reflect this periodicity. With evidence for climatic warming in some regions of Antarctica, there is concern that ecosystem change may be induced by changes in sea-ice extent. The relative abundance of krill and salps appears to change interannually with sea-ice extent, and in warm years, when salps proliferate, krill are scarce and dependent predators suffer severely. Further research on the Southern Ocean sea-ice system is required, not only to further our basic understanding of the ecology, but also to provide ecosystem managers with the information necessary for the development of strategies in response to short- and medium-term environmental changes in Antarctica. Technological advances are delivering new sampling platforms such as autonomous underwater vehicles that are improving vastly our ability to sample the Antarctic under sea-ice environment. Data from such platforms will enhance greatly our understanding of the globally important Southern Ocean sea-ice ecosystem.

Animals↗
PubMed2000

Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands.

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.

Animals↗
PubMed2002

The effect of anions on azide binding to myoglobin: an unusual functional modulation.

The effect of increasing concentrations of several anions on the azide (N(-)(3)) binding properties of sperm whale and horse ferric myoglobin has been studied. Surprisingly, a number of anions may act as heterotropic effectors, decreasing the affinity of myoglobins for N(-)(3), in the following order: ClO(-)(4)=I(-)>Br(-)>Cl(-) and SO(2-)(4), which mirrors the increase in their charge density. The largest effects were measured using ClO(-)(4) and I(-), which produce a 4-fold and 8-fold reduction of the N(-)(3) binding affinity in horse and sperm whale myoglobins, respectively. A dissociation equilibrium constant (K(d)) ranging from 150 to 250 mM was estimated for ClO(-)(4) and I(-) binding to myoglobins. In order to analyse the molecular mechanism producing the reduction of the N(-)(3) binding affinity to ferric myoglobin, the potential anionic binding sites within ferric myoglobin were investigated by a molecular modelling study using the program Grid. Analysis of the theoretical results suggests two particularly favourable binding sites: the first, next to the distal side of the haem, whose occupancy might alter the electrostatic potential surrounding the bound N(-)(3); the second, involving residues of helices B and G which are far from the haem iron atom, thus implying a long range effect on the bound N(-)(3). Based on the evidence that no significant conformational changes are found in the three-dimensional structures of N(-)(3)-free and N(-)(3)-bound myoglobin and on previous results on N(-)(3) binding to ferric myoglobin mutants in CD3 positions, we favour the first hypothesis, suggesting that the functional heterotropic modulation of monomeric myoglobin is mainly depending on a decrease of the positive charge density induced by the binding of anions to the haem distal side.

Allosteric Regulation↗
PubMed2003

Trace elements in two odontocete species (Kogia breviceps and Globicephala macrorhynchus) stranded in New Caledonia (South Pacific).

Liver, muscle and blubber tissues of two short-finned pilot whales (Globicephala macrorhynchus) and two pygmy sperm whales(Kogia breviceps) stranded on the coast of New Caledonia have been analysed for 12 trace elements (Al, Cd, Co, Cr, Cu. Fe, organic and total Hg, Mn, Ni, Se, V, and Zn). Liver was shown to be the most important accumulating organ for Cd, Cu, Fe, Hg, Se, and Zn in both species, G. macrorhynchus having the highest Cd, Hg, Se and Zn levels. In this species, concentrations of total Hg are particularly elevated, reaching up to 1452 microg g(-1) dry wt. Only a very low percentage of the total Hg was organic. In both species,the levels of Hg are directly related to Se in liver. Thus, a molar ratio of Hg:Se close to 1.0 was found for all specimens, except for the youngest K. breviceps. Our results suggest that G. macrorhynchus have a physiology promoting the accumulation of high levels of naturally occurring toxic elements. Furthermore, concentrations of Ni, Cr and Co are close to or below the detection limit in the liver and muscles of all specimens. This suggests that mining activity in New Caledonia, which typically elevates the levels of these contaminants in the marine environment, does not seem to be a significant source of contamination for these pelagic marine mammals.

Adipose Tissue↗
PubMed2003

Trophic transfer of persistent organochlorine contaminants (OCs) within an Arctic marine food web from the southern Beaufort-Chukchi Seas.

Stable isotope values (13C, 15N) and concentrations of persistent organochlorine contaminants (OCs) were determined to evaluate the near-shore marine trophic status of biota and biomagnification of OCs from the southern Beaufort-Chukchi Seas (1999-2000) near Barrow, AK. The biota examined included zooplankton (Calanus spp.), fish species such as arctic cod (Boreogadus saida), arctic char (Salvelinus alpinus), pink salmon (Oncorhynchus gorbuscha), and fourhorn sculpin (Myoxocephalus quadricornis), along with marine mammals, including bowhead whales (Balaena mysticetus), beluga whales (Delphinapterus leucas), ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus). The isotopically derived trophic position of biota from the Beaufort-Chukchi Seas marine food web, avian fauna excluded, is similar to other coastal food webs in the Arctic. Concentrations of OCs in marine mammals were significantly greater than in fish and corresponded with determined trophic level. In general, OCs with the greatest food web magnification factors (FWMFs) were those either formed due to biotransformation (e.g. p,p'-DDE, oxychlordane) or considered recalcitrant (e.g. -HCH, 2,4,5-Cl substituted PCBs) in most biota, whereas concentrations of OCs that are considered to be readily eliminated (e.g. -HCH) did not correlate with trophic level. Differences in physical-chemical properties of OCs, feeding strategy and possible biotransformation were reflected in the variable biomagnification between fish and marine mammals. The FWMFs in the Beaufort-Chukchi Seas region were consistent with reported values in the Canadian Arctic and temperate food webs, but were statistically different than FWMFs from the Barents and White Seas, indicating that the spatial variability of OC contamination in top-level marine Arctic predators is attributed to differences in regional sources of contamination rather than trophic position.

Animals↗
PubMed1998

The use of microsomal in vitro assay to study phase I biotransformation of chlorobornanes (Toxaphene) in marine mammals and birds. Possible consequences of biotransformation for bioaccumulation and genotoxicity.

The factors determining the bioaccumulation of lipophilic compounds in wildlife are often poorly understood, partly because it is difficult to do in vivo experiments with animals such as marine mammals and birds. To evaluate the role of phase I biotransformation in the bioaccumulation process of chlorobornanes (toxaphene), this was studied in in vitro assays with hepatic microsomes of animals that could be sampled shortly after death. The capacity of microsomes to metabolise a technical toxaphene mixture decreased in the order Phoca vitulina (harbour seal) >> Lagenorhynchus albirostris (whitebeaked dolphin) approximately equal to Diomedea immutabilis (Laysan albatross) > Physeter macrocephalus (sperm whale). Harbour seal microsomes metabolised the chlorobornane (CHB) congeners CHB-32 and CHB-62; whitebeaked dolphin and Laysan albatross microsomes only metabolised CHB-32. Metabolism of CHB-26 and CHB-50 was never observed. The negative chemical ionisation (NCI-) mass spectra of some of the hydroxylated metabolites were obtained. The number of peaks in the toxaphene residues of wildlife extracts decreased in the order of increasing in-vitro biotransformation capacity. Thus, the results of the in vitro assays and residue analysis were in accordance, although assays with microsomes of more individuals of the same species are required for a more general conclusion at the species level. Finally, the effect of in vitro biotransformation was evaluated in terms of the genotoxic potential using the Mutatox assay. Only technical toxaphene and CHB-32 were genotoxic in the direct assay, whereas the addition of rat S9 fraction or microsomes of harbour seal and albatross decreased the genotoxic response. Thus, organisms with a low ability to metabolise chlorobornanes, such as whales, may be most affected by the carcinogenic properties of toxaphene. A hypothetical reaction which fits the experimental results is discussed. Based on these results it is concluded that in vitro assays with microsomes of wildlife animals which died a natural cause can act as a valuable tool to assess the occurrence and effects of phase I metabolism. Some precautions are discussed, that should be taken to reduce the chance of false negative results.

Animals↗
PubMed2003

Construction and characterization of a chimeric myoglobin.

In order to investigate the functional and structural role of modular structure in globins, we have engineered a chimeric myoglobin (ChimMb) in which the first and third exon come from the gene coding for the sperm whale Mb and the second exon from the gene coding for Aplysia limacina Mb. This ChimMb, fused to the Maltose Binding Protein (MBP) and expressed in Escherichia coli as an apoprotein, binds protoheme in a 1:1 stoichiometric ratio. Based on some functional and spectroscopic properties, we conclude that the central core of the ChimMb (which derives from A. limacina) is native-like. On the other hand, the ChimMb deprived (by proteolytic digestion) of the fused MBP displays a considerably reduced stability. These results suggest that the sperm whale A-G-H nucleus does not contribute significantly to the overall stability of the ChimMb.

Amino Acid Sequence↗
PubMed1994

Stability of myoglobin: a model for the folding of heme proteins.

Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells. The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission. Two key conclusions were reached. First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa. In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression. Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin. Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant. In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin. The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation.

Animals↗
PubMed1984

Conformational differences between various myoglobin ligated states as monitored by 1H NMR spectroscopy.

In paramagnetic metmyoglobin, cyanomyoglobin (CNMb), and deoxymyoglobin, His-36 has a high pK (approximately 8), and the NMR titration behavior of the H-2 resonance is perturbed, due to the presence at low pH of a hydrogen bond with Glu-38, which is broken at high pH. The His-36 H-4 resonance shows no shift with pK approximately 8 because of two opposing chemical shift effects but monitors the titration of nearby Glu-36 (pK = 5.6). In diamagnetic derivatives [(carbon monoxy)myoglobin (COMb) and oxymyoglobin (oxyMb)], the titration behavior of His-36 H-2 and H-4 resonances is normalized (pK approximately 6.8). The very slight alkaline Bohr effect in sperm whale myoglobin (Mb) is interpreted in terms of the pK change of His-36 from deoxyMb to oxyMb and compensating pK changes in the opposite direction of other unspecified groups. In sperm whale COMb at 40 degrees C, the distal histidine (His-64) and His-97 have pK values of 5.0 and 5.9. The meso proton resonances remote from these groups do not show a titration shift, but the nearby gamma-meso proton (pK = 5.3) responds to titration of both histidines, and the upfield Val-68 methyl at -2.3 ppm (pK = 4.7) witnesses the titration of nearby His-64. At 20 degrees C, the latter resonance is reduced in size, and a second resonance occurs at -2.8 ppm, which is insensitive to pH and, hence, more remote from His-64. Both resonances arise from two conformations of Val-68 in slow equilibrium. In oxyMb at 20 degrees C, only the latter resonance is observed, presumably because of the steric restrictions imposed by the hydrogen bond between ligand and His-64 in oxyMb, which is not present in COMb. In oxyMb the pK of His-97 (5.6) is similar to that of the meso proton resonances (5.5) and to the pK of other pH-dependent processes, including the very small acid Bohr effect. It is likely that these processes are controlled by the titration of His-97.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements.

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗