Carp Hemoglobin Research Articles

The effect of conformational changes on non‐coordinated water occupancy associated with R and T allosteric states of Carp Hemoglobin

The chemistry of non‐coordinated water molecules in the apolar internal environment of proteins is poorly understood. Our recent discovery of a spectral marker for the entry of noncoordinated water into the distal heme pocket of myoglobin has helped clarify this molecule's functional importance in ligand binding. We will apply our novel spectrokinetic assay to test the hypothesis that conformational changes can affect ligand binding indirectly by changing the occupancy of non‐coordinated water in apolar cavities. A comparison of the spectrokinetics between the visible and Soret spectral regions distinguishes protein relaxations from those associated with changes in internal water occupancy. Hemoglobin from Carp, Cyprinus carpio, shows differences in the putative water occupancy signal between the R and T allosteric states, which correspond to changes in ligand binding dynamics. These results suggest that conformational induced changes in water occupancy may play a role in allosteric induced changes in ligand binding dynamics. This knowledge will increase the understanding of the role these water molecules play in allosteric regulation.

Structural basis for the specific interaction of chicken haemoglobin with bromophenol blue: a computational analysis

It has been observed that bromophenol blue interacted specifically with chicken haemoglobin but not with carp haemoglobin during electrophoresis, but the mechanism of interaction is still not well understood. In this computational study, the binding of bromophenol blue to chicken haemoglobin has been investigated using sequence alignment, homology modelling, electrostatic potential distribution and flexible docking methods. Molecular modelling studies reveal that bromophenol blue-binding site, formed by residues Val1α, Leu2α, Ala131α, Thr134α, Ala138α and Arg141α, is located between two α chains of chicken haemoglobin, and the binding is dominated by hydrophobic interactions. Moreover, comparison of chicken and carp haemoglobin structural models provides a structural rationale for the recognition of bromophenol blue by chicken haemoglobin. These principles can in turn be used to study the molecular recognition mechanism and design a mimic of bromophenol blue for the development of new haemoglobin binders.

Nitric oxide formation from the reaction of nitrite with carp and rabbit hemoglobin at intermediate oxygen saturations

The nitrite reductase activity of deoxyhemoglobin has received much recent interest because the nitric oxide produced in this reaction may participate in blood flow regulation during hypoxia. The present study used spectral deconvolution to characterize the reaction of nitrite with carp and rabbit hemoglobin at different constant oxygen tensions that generate the full range of physiological relevant oxygen saturations. Carp is a hypoxia-tolerant species with very high hemoglobin oxygen affinity, and the high R-state character and low redox potential of the hemoglobin is hypothesized to promote NO generation from nitrite. The reaction of nitrite with deoxyhemoglobin leads to a 1 : 1 formation of nitrosylhemoglobin and methemoglobin in both species. At intermediate oxygen saturations, the reaction with deoxyhemoglobin is clearly favored over that with oxyhemoglobin, and the oxyhemoglobin reaction and its autocatalysis are inhibited by nitrosylhemoglobin from the deoxyhemoglobin reaction. The production of NO and nitrosylhemoglobin is faster and higher in carp hemoglobin with high O(2) affinity than in rabbit hemoglobin with lower O(2) affinity, and it correlates inversely with oxygen saturation. In carp, NO formation remains substantial even at high oxygen saturations. When oxygen affinity is decreased by T-state stabilization of carp hemoglobin with ATP, the reaction rates decrease and NO production is lowered, but the deoxyhemoglobin reaction continues to dominate. The data show that the reaction of nitrite with hemoglobin is dynamically influenced by oxygen affinity and the allosteric equilibrium between the T and R states, and that a high O(2) affinity increases the nitrite reductase capability of hemoglobin.

Nitro musk metabolites bound to carp hemoglobin: determination by GC with two MS detection modes: EIMS versus electron capture negative ion MS

Nitroaromatic compounds including synthetic nitro musks are important raw materials and intermediates in the synthesis of explosives, dyes, pesticides, and pharmaceutical and personal-care products (PPCPs). The nitro musks such as musk xylene (MX) and musk ketone (MK) are extensively used as fragrance ingredients in PPCPs and other commercial toiletries. Identification and quantification of a bound 4-amino-MX (4-AMX) metabolite as well as a 2-amino-MK (2-AMK) metabolite were carried out by gas chromatography–mass spectrometry (GCMS), with selected ion monitoring (SIM) in both the electron ionization (EIMS) and electron capture (EC) negative ion chemical ionization (NICIMS) modes. Detection of 4-AMX and 2-AMK occurred after the cysteine adducts in carp hemoglobin, derived from the nitroso metabolites, were released by alkaline hydrolysis. The released metabolites were extracted into n-hexane. The extract was preconcentrated by evaporation, and analyzed by GC-SIM-MS. A comparison between the EI and EC approaches was made. EC NICIMS detected both metabolites whereas only 4-AMX was detected by EIMS. The EC NICIMS approach exhibited fewer matrix responses and provided a lower detection limit. Quantitation in both approaches was based on an internal standard and a calibration plot.

Determination of a Bound Musk Xylene Metabolite in Carp Hemoglobin as a Biomarker of Exposure by Gas Chromatography-Mass Spectrometry Using Selected Ion Monitoring

Musk xylene (MX) is widely used as a fragrance ingredient in commercial toiletries. Identification and quantitation of a bound 4-amino-MX (AMX) metabolite was carried out by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). Detection of AMX occurred after the cysteine adducts in carp hemoglobin (Hb), derived from the nitroso metabolite, were released by alkaline hydrolysis. The released AMX metabolite was extracted into n-hexane. The extract was preconcentrated by evaporation and analyzed by GC-SIM-MS. The concentration of AMX metabolite was found to range from 6.0 to 30.6 ng/g in the carp Hb, collected from the Las Vegas Wash and Lake Mead, NV areas. The presence of an AMX metabolite in the carp Hb was confirmed when similar mass spectral features and the same retention time of the AMX metabolite were obtained for both standard AMX and carp Hb extract solutions. In the nonhydrolyzed and reagent blank extracts, the AMX metabolite was not detected.

Protein relaxation without a geminate phase in nanosecond photodissociated CO carp hemoglobin

Transient heme–protein interactions upon passing from ligated to deligated carp hemoglobin were observed through time-resolved optical spectra following nanosecond CO photodissociation. The spectral evolution of the heme, in the nanosecond and microsecond time ranges, shows a protein conformational relaxation and the absence of a geminate CO recombination in carp hemoglobin. The comparison of the phenomena in carp and human hemoglobin implies that the physical basis of the geminate rebinding in human hemoglobin should involve an out-of-equilibrium protein conformation, close to a dissipative structure defined by the thermodynamics of Prigogine.

Cloning and sequencing of cDNAs of the β-globin gene family in carp.

A total blood cell cDNA library was constructed using a 3-year-old carp Cyprinus carpio. A β-globin cDNA (CβG1) was identified from the library by the polymerase chain reaction using a β-globin-specific primer deduced from the carp β-globin-A amino acid sequence. Also, five additional types of β-globin cDNAs (CβG2∼6) were isolated by colony hybridization using CβG1 as a probe. Sequence analysis revealed that these CβGs encoded 147 amino acids, and the deduced amino acid sequences showed high identity (89·1-95·2%) to previously reported carp β-globin amino acid sequences. The nucleotide sequences of the CβGs were very similar (identity 96·0-99·6%) and the expression levels of CβG1∼6 were 28·6, 28·6, 21·4, 14·3, 3·6 and 3·6% of the total number of cloned CβGs, respectively. Although the complete amino acid sequence identities between the CβGs and the β-globin of higher vertebrates were low, functionally important regions such as the α-βcontact region and haem contact region were well conserved. These data showed that, as in higher vertebrates, the adult carp has a multiple β-globin gene family (at least six members). However, transcripts encoding four types of peptides (CβG1 type, CβG2 and 3 type, CβG5 type, and CβG4 and 6 type) were expressed at relatively high levels, this being a unique character of the carp haemoglobin system.

Isolation and Stability of Partially Oxidized Intermediates of Carp Hemoglobin: Kinetics of CO Binding to the Mono- and Triferric Species

The monoliganded and triliganded forms of the asymmetric valency hybrids of carp hemoglobin were isolated using high-performance liquid chromatography. These partially oxidized hybrids were shown to be sufficiently stable to permit the measurement of the kinetics of CO binding. The effects of protons and inositol hexaphosphate on the rates of these reactions were examined. The kinetics of CO recombination with these partially oxidized derivatives were compared to the kinetics of CO binding to the fully ferrous molecule. To a first approximation, the kinetic behavior of the monoferric derivative was consistent with a small shift in the T<==>R equilibrium in favor of the R state. The presence of three ferric ligands resulted in a still greater shift in the conformational equilibrium in favor of the R state. The kinetic behavior of the triferric molecule was similar, but not identical, to that of a fully ferrous molecule which is triliganded with CO. The properties of both asymmetric valency hybrids were responsive to the nature of the ligand; i.e., the rate of CO binding was increased more by the presence of cyanide on the ferric hemes than by water. Not all of the data could be accommodated within the two-state model. For example, there was evidence of an altered T state in the case of the tricyanomet derivative at low pH in the presence of inositol hexaphosphate.

The Effect of 75% Glycerol on the Oxygen-Binding Properties of Carp Hemoglobin

At pH 6 in the presence of inositol hexaphosphate, IHP, conditions where ligand-saturated carp hemoglobin is already in the low affinity T state, the addition of glycerol has little effect on affinity and ligand binding remains noncooperative. At all other pH values examined, with and without IHP, the effect of glycerol is to lower oxygen affinity possibly by shifting the equilibrium between the T state and the high affinity R state in the direction of the T state. Although glycerol does not appear to have an appreciable effect on the T state itself, a small effect on the R state cannot be excluded by our data.

Selective examination of heme protein azide ligand-distal globin interactions by vibrational circular dichroism

Vibrational circular dichroism (VCD) spectra of the antisymmetric stretch of azide ligated to the heme of a series of evolutionarily diverse and site-directed mutant hemoglobins and myoglobins are anomalously intense and demonstrate an intriguing sensitivity to subtle protein-ligand interactions. The antisymmetric stretch of the azide ligand covalently bound to the low-spin iron shows an anisotropy ratio of -9.5 X 104 for sperm whale and horse myoglobin which decreases to -8.0 X 104 for human and carp hemoglobin and Chironimus thummi thummi III monomeric hemoglobin. The VCD spectra of these heme-azide complexes depend upon the interactions of the azide ligand with distal heme pocket residues such as the E7 distal histidine and El 1 valine. The site-directed mutants of sperm whale (distal histidine substituted GIy E7) and human (distal valine substituted Asn El 1) myoglobin have vanishingly small anisotropy ratios (<-0.5 X 1(T 4), while elephant myoglobin (distal histidine substituted GIn E7) shows an anisotropy ratio of-6.4 X ICT 4. The azide ligand ionically bound to a high-spin iron shows a vanishingly small VCD intensity.

Quaternary structure and the geminate recombination of carp hemoglobin with methylisocyanide

The kinetics of geminate recombination were studied for the methylisocyanide derivative of carp hemoglobin. Carp hemoglobin is of interest because it has been established that the fully liganded form switches between a high affinity R state at pH 9 and a low affinity T state at pH 6 in the presence of IHP. Geminate recombination was observed on both the picosecond and the nanosecond time scales under all conditions; however, only a small variation is observed in the rates and the yields of geminate recombination as the protein switches from the R to the T state. Taken together with overall “on” and “off” rates, the data indicate that the change from the R to the T configuration affects bond breaking most, but also influences subsequent escape from the protein as well as both entry into the protein and bond formation. There is some reason to postulate tertiary conformational change in the T state on the microsecond time scale following ligand escape from the protein.

Spin equilibria in human methemoglobin: effects of bezafibrate and inositol hexaphosphate as measured by susceptometry and visible spectroscopy

The effects of inositol hexaphosphate (IHP) and a second allosteric effector, bezafibrate, on the spin-state equilibria of the mixed-spin derivatives of ferric human hemoglobin A are examined. Changes in spin-state equilibrium are monitored by measuring absorption spectra in the visible region (460-700 nm) as well as by direct measurements of magnetic susceptibility by means of a superconducting fluxmeter. The addition of IHP at pH 6.5 results in a measurable shift in the spin equilibria of these derivatives toward higher spin. However, the addition of bezafibrate in the presence of IHP results in still larger shifts toward the high-spin form. The changes in the free energies of the spin-state equilibria resulting from the combination of these two effectors are similar in magnitude to that which results from the R-state to T-state transition in carp hemoglobin.

Interaction of allosteric effectors (ATP, CO2, H+) modulating oxygen affinity of the hemoglobin in the carp,Cyprinus carpio, in vitro

The interaction of allosteric effectors (CO2, ATP, H+) with respect to the oxygen affinity of carp hemoglobin was analyzed by determining oxygen binding curves spectrophotometrically in dilute solutions of stripped hemoglobin at 20°C. The pH range studied was 6.8–8.2.P CO2 was 0, 10 and 70 mmHg (0, 1.33 and 9.3 kPa). ATP/Hb4 was 0, 8 and 24. In the presence of either CO2 or ATP, the effects of the cofactors onP 50 were as expected over the whole pH range. In contrast to other published data, each cofactor also had a significant effect onP 50 in the presence of the other cofactor. Evidence was obtained that oxylabile carbamate is formed by carp hemoglobin and that the formation of carbamate persists at a lower level in the presence of ATP. The results support the view that the binding of ATP to carp hemoglobin requires only one terminal amino group, leaving the other N-terminal of the β-chain free to react with CO2.

Anion transport, chloride cell number and nitrite-induced methaemoglobinaemia in rainbow trout ( Salmo gairdneri) and carp ( Cyprinus carpio)

The toxic effects of nitrite on ionic transport and haemoglobin function in rainbow trout ( Salmo gairdneri Richardson) and carp ( Cyprinus carpio L.) were investigated. Under similar environmental conditions the maximal rates of chloride and nitrite uptake in carp (25 and 81 μmol h −1kg −1) were lower than in rainbow trout (368 and 198 μmol h −1kg −1). Carp possess 47% less gill chloride cells than trout and the relationship between anion transport and the chloride cell population is discussed. In vitro, methaemoglobin formation occurs more rapidly in carp blood, 5.58% h −1 compared with trout, 3.39% h −1. The carp haemoglobin molecule is more sensitive to nitrite oxidation than trout haemoglobin and the rate of methaemoglobin reduction is more rapid in carp red cells. The principal reason for carp being more tolerant to nitrite than are trout is because of the lower rate of branchial uptake of nitrite, rather than because of any effects on haemoglobin.

Comparison of models for reassociation of carbon monoxide with carp haemoglobin

A comparison among three models for reassociation of carbon monoxide with carp haemoglobin, in relaxed state, is made for temperatures below 200 K. The experimental data utilized are from Cobau et al. who analysed them according to one of these models. The differences among the models lead to distinct shapes of their energy distributions. Nevertheless, it was verified that the peak energy of these distributions and the Arrhenius frequency factor are independent of the models. Their values are smaller for haemoglobin than for myoglobin and the distribution is wider for myoglobin, showing that it has a larger number of conformational substates. The three models studied reproduce well the experimental curves of 100, 140 and 180 K.

Effects of ligand size on pH and organic phosphate-dependent affinity changes in carp hemoglobin as measured by isonitrile binding

The equilibria of the binding of methyl and ethyl isonitrile to carp hemoglobin have been measured at three pH values in the presence and absence of inositol hexaphosphate. The binding of methyl isonitrile is characterized by a higher overall dissociation constant, C 1 2 , and a higher Hill coefficient, n, than that of the ethyl derivative. The former is consistent with the greater hydrophobicity of ethyl isonitrile, and the latter is probably due to a greater intrinsic difference or heterogeneity in the binding affinities of the α- and β-chains for the larger ligand. Changes in log C 1 2 which result from alterations in pH or addition of organic phosphate are the same for both ligands within experimental error. This result is not consistent with affinity changes being the result of steric interactions between the protein and the ligand. At pH 6 in the presence of inositol hexaphosphate, equilibrium parameters estimated from overall rates of ligand binding and dissociation are in good agreement with direct equilibrium measurements. This is consistent with the protein being in a low-affinity, T-like state even when saturated with ligand under these conditions, resulting in a loss of cooperativity in ligand binding. At high pH, ligand binding remains cooperative, as evidenced by n values greater than unity, a general lack of agreement between measured equilibrium parameters and those estimated from overall kinetic constants, and differences in the kinetics of ligand binding as observed by rapid-mixing and flash photolysis techniques. Thus, the deoxygenated state of carp hemoglobin at high pH does not appear to be a good model of a deoxygenated R quaternary structural state.

Studies on the linkage between spin equilibria and protein structure in carp ferric hemoglobin

The effects of protein conformation on the spin-state equilibria of several derivatives of carp hemoglobin have been examined. This has been done by measuring the pH dependence of the paramagnetic susceptibilities of these derivatives in the presence and absence of inositol hexakisphosphate, P6-inositol. In all cases the addition of P6-inositol at low pH and the lowering of the pH in the presence of P6-inositol shift the spin-state equilibrium in favor of the high-spin electronic configuration. The P6-inositol and pH dependence of these magnetic properties parallels the pH and P6-inositol dependence of the conformational state of the hemoglobin as determined in earlier studies and further supports a thermodynamic linkage between the electronic state of the iron atoms and the quaternary structure of the hemoglobin molecule.

Intrinsic fluorescence of carp hemoglobin: a study of the R → T transition

The intrinsic fluorescence of hemoglobins is known to respond to ligand-induced changes in the quaternary structure of the protein. Carp hemoglobin is an interesting model to study the quaternary transition since its R ↔ T equilibrium is pH-dependent and at low pH, in the presence of organic phosphate, it remains in the T or ‘deoxy’ quaternary structure, even when saturated with ligand. In this study, using front-face fluorometry, we show that the intrinsic fluorescence intensity exhibited by carp carboxyhemoglobin increases as the pH is lowered below 6.5 in the presence of inositol hexaphosphate. At low pH, carp methemoglobin is less affected by the addition of inositol hexaphosphate than is the CO derivative, while little or no change is observed in the met-azide derivative. We conclude: (1) the exact nature of the R to T state transition induced by inositol hexaphosphate differs for carp carboxy-, met- and met-azide hemoglobin derivatives; (2) the chromophores responsible for the changes observed with absorption spectroscopy may not be the same as those chromophores responsible for the fluorescence differences; and (3) α46-Trp is tentatively assigned as one source of fluorescence emission. Furthermore, fluorescence properties of carp hemoglobin are compared to those of human hemoglobin.

XANES spectroscopy of carp hemoglobin-iron in correlation with the affinity changes of the protein for ligand

The strong variation of ligand-binding properties with pH for carp hemoglobin is not reflected in the electronic distribution of the heme-iron. Thus, we can suppose that hemoglobin affinity is directly controlled by the protein and not by some particular changes of the iron atom.

Analysis of zeros of binding polynomials for tetrameric hemoglobins.

A quantitative measure of the validity of the MWC description of cooperative binding equilibria has been obtained which uses only the Adair constants. This is accomplished through simple relationships using the zeros of the Adair binding polynomial and unique properties of the zeros of MWC polynomials as described in the accompanying paper (W.E. Briggs, Biophys. Chem. 24 (1986) 311). The method is applied to oxygen binding to a large number of hemoglobins under a wide variety of conditions. In most cases, exemplified by human hemoglobin under a wide range of conditions, the MWC model is allowed and the probability of its suitability is determined. The probability given by this method correlates directly with the deviation between the experimental binding curve and that derived from the theory. In several cases the pattern of the Adair polynomial zeros immediately excludes the MWC model, most notably for carp hemoglobins. A physical picture of cooperative binding site interactions is nevertheless obtained from the patterns of zeros as they relate to the factorization of the binding polynomial.