Hydration-dependent conformational states of hemoglobin. Equilibrium and kinetic behavior

The equilibrium and kinetics of methemoglobin conversion to hemichrome induced by dehydration were investigated by visible absorption spectroscopy. Below about 0.20 g water per g hemoglobin only hemichrome was present in the sample; above this value, an increasing proportion of methemoglobin appeared with the increase in hydration. The transition between the two derivatives showed a time-dependent biphasic behavior and was observed to be reversible. The rates obtained for the transition of methemoglobin to hemichrome were 0.31 and 1.93 min-1 and for hemichrome to methemoglobin 0.05 and 0.47 min-1. We suggest that hemichrome is a reversible conformational state of hemoglobin and that the two rates observed for the transition between the two derivatives reflect the alpha- and beta-chains of hemoglobin.     

Solid state fluorescence of lyophilized proteins

Fluorescence spectroscopy has been used to measure changes in the tertiary structure of proteins in the solution state. The sensitivity of fluorescence to the protein tryptophan environment has made it a useful tool for studying protein conformation and stability. Using fluorescence spectroscopy to probe structural alterations in lyophilized proteins has been limited due to technical challenges and overwhelming background light scattering. We have investigated the possibility of analyzing lyophilized proteins using the Cary-Eclipse spectrofluorometer by monitoring the fluorescence of the protein therapeutic after subjecting the lyophilized cake to heat-induced accelerated degradation. We have been able to obtain reproducible fluorescence spectra, detecting possible structural changes under these conditions. Fluorescence and circular dichroism spectroscopic analyses of the reconstituted proteins indicated that changes in fluorescence intensities observed in the solid state could be correlated to that in solution and to possible tertiary structural changes. Size exclusion chromatography analysis of protein Y subject to accelerated degradation showed a correlation between decreasing fluorescence intensity and increasing protein Y tetramer in solution, consistent with long-term stability. This suggests that solid state, intrinsic protein fluorescence measurements using the Cary-Eclipse holder may be feasible for long-term stability studies and formulation development.     

Proton NMR study of methemoglobin and its isolated chains. Effect of the subunit association on the structure of the subunits.

In order to investigate the effect of the alpha beta subunit contacts on the subunit structure of human adult methemoglobin, the hyperfine shifted proton NMR spectra of several high spin complexes (water, cyanate, thiocyanate, formate, fluoride, and nitrite) and low spin complexes (imisazole, azide, and cyanide) of hemoglobin and its isolated subunits were characterized at 220 MHz and 22 degrees C. The spectra of ferric low spin derivatives of the isolated subunits were approximately superimposable on the corresponding hemoglobin spectra. On the other hand, the high spin spectra of the isolated subunits were greatly different from each other. The spectral anomaly in the ferric high spin complexes of the isolated beta subunit were interpreted to indicate other structural change than the hemichrome formation in the beta heme pocket. Difference in the subunit association effect between the high and low spin complexes of the isolated beta subunit was interpreted on the basis of a conformational change of the apoprotein dependent on the spin state of the beta heme iron.     

GFT projection NMR spectroscopy for proteins in the solid state

Recording of four-dimensional (4D) spectra for proteins in the solid state has opened new avenues to obtain virtually complete resonance assignments and three-dimensional (3D) structures of proteins. As in solution state NMR, the sampling of three indirect dimensions leads per se to long minimal measurement time. Furthermore, artifact suppression in solid state NMR relies primarily on radio-frequency pulse phase cycling. For an n-step phase cycle, the minimal measurement times of both 3D and 4D spectra are increased n times. To tackle the associated ‘sampling problem’ and to avoid sampling limited data acquisition, solid state G-Matrix Fourier Transform (SS GFT) projection NMR is introduced to rapidly acquire 3D and 4D spectral information. Specifically, (4,3)D (HA)CANCOCX and (3,2)D (HACA)NCOCX were implemented and recorded for the 6 kDa protein GB1 within about 10% of the time required for acquiring the conventional congeners with the same maximal evolution times and spectral widths in the indirect dimensions. Spectral analysis was complemented by comparative analysis of expected spectral congestion in conventional and GFT NMR experiments, demonstrating that high spectral resolution of the GFT NMR experiments enables one to efficiently obtain nearly complete resonance assignments even for large proteins.     

The oxidation process of Antarctic fish hemoglobins.

Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation process, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectroscopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemoglobins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemichrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly transformed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquomet and the hemichrome. In order to assign the heme region state of the alpha and beta chains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an alpha(aquomet)-beta(bishistidyl-hemichrome) state. This demonstrates that the alpha and beta chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrome state [Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A. & Mazzarella, L. (2002) Proc. Natl Acad. Sci. USA99, 9801-9806], the quaternary structures of these alpha(aquomet)-beta(bishistidyl-hemichrome) forms are intermediate between the physiological R and T hemoglobin states. Together, these structures provide information on the general features of this intermediate state.     

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H₂O₂, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H₂O₂ or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.     

Thermal stability and conformational structure of salmon calcitonin in the solid and liquid states

Salmon calcitonin (sCT) was selected as a model protein drug for investigating its intrinsic thermal stability and conformational structure in the solid and liquid states by using a Fourier transform infrared (FT‐IR) microspectroscopy with or without utilizing thermal analyzer. The spectral correlation coefficient (r) analysis between two second‐derivative IR spectra was applied to quantitatively estimate the structural similarity of sCT in the solid state before and after different treatments. The thermal FT‐IR microspectroscopic data clearly evidenced that sCT in the solid state was not effected by temperature and had a thermal reversible property during heating–cooling process. Moreover, the high r value of 0.973 or 0.988 also evidenced the structural similarity of solid‐state sCT samples before and after treatments. However, sCT in H₂O exhibited protein instability and thermal irreversibility after incubation at 40°C. The temperature‐induced conformational changes of sCT in H₂O was occurred to transform the α‐helix/random coil structures to β‐sheet structure and also resulted in the formation of intramolecular and intermolecular β‐sheet structures. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 200–207, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Properties of trout hemoglobin covalently bound to a solid matrix.

This paper reports the ligand binding properties of the major hemoglobin component from trout (Salmo irideus) covalently bound to a solid matrix (Sepharose or Sephadex). A comparison between the functional properties of this protein in solution and of the protein-matrix complex shows significant changes although the basic properties of the molecule are maintained on covalent binding to Sepharose (or Sephadex). Thus the Root effect, characteristic of Hb trout IV, is still present while the heme-heme interactions are, on the average, smaller in the matrix bound protein as compared to the soluble form. No differences in the O2 binding properties were observed when the protein was coupled to the resin, as the ligand bound or as the ligand free derivative. Although an unequivocal interpretation of the data is made difficult by the lack of information on the number and identity of the groups involved in the coupling, the main changes in the protein functional properties may be related to the chemical modifications "per se" more than to the immobilization imposed to the macromolecule by coupling to the matrix. Structural changes which mainly involve perturbation of the tertiary structure of the molecule may qualitatively rationalize the data.     

Acid-Precipitated M Protein Compared with a Column-Eluted M Protein Preparation from Type 12, Group A Streptococci

An M protein preparation of group A streptococci, precipitated with 0.03 m sodium acetate buffer (pH 4.0) was compared with a column-eluted M protein preparation. Absorption spectra and methyl pentose content were similar in both preparations. Acrylamide gel electrophoresis patterns were different. Gel diffusion demonstrated two lines of fusion in the preparations. More antigens could be demonstrated in both preparations by using immunoelectrophoresis. Neither the pH 4 precipitate nor the column-eluted preparation appeared to be a pure M protein preparation.     

Conformation of alpha zeins in solid state by Fourier transform IR

The major maize storage proteins (alpha zeins) are deposited as an insoluble mass in the protein bodies of the endosperm. Because they are insoluble in water, most structural studies are performed in alcohol solutions. To solve the question raised by several authors about denaturation of the alpha zein structure by alcohol, we analyze the secondary structure of alpha zeins prepared with and without solubilization in alcohol (corn gluten meal and protein bodies with high concentrations of alpha zeins and traces of beta zeins). The secondary structures of alpha zeins are analyzed in the solid state by Fourier transform IR spectroscopy (FTIR) in KBr pellets and solid-state 13C-NMR spectroscopy. The proportion of secondary structures obtained by FTIR of alpha zeins prepared with and without solubilization in alcohol yield almost identical proportions of alpha helices and beta sheets. The proportion of alpha helices (43%) agrees with that measured by circular dichroism in an alcohol solution. However, the proportion of beta sheets (28%) is higher than the one measured by the same technique. Gluten and protein body samples with high beta zein content showed higher beta sheet and lower alpha helix proportions than that obtained for alpha zein preparations. The solid-state 13C-NMR spectra show the carbonyl peak for the alpha zeins at delta 176 and for the sample rich in beta zeins at delta 172, which demonstrates the presence of a high content of alpha helices and beta sheets, respectively. These results indicate that alcohol solubilization does not affect the conformation of alpha zeins, validating the secondary structure measurements in solution.     

pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase.

The spectral (e.p.r. and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined. At neutral pH the e.p.r. and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively. By lowering pH, the e.p.r. and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms. By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond. However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant.     

Oxidation and denaturation of hemoglobin encapsulated in liposomes

A study was made of the in vitro stability of hemoglobin-containing liposomes (‘hemosomes’) prepared from phosphatidylcholines, equimolar cholesterol and red cell lysate by the hand-shaking and ether-injection methods. Absorption spectra indicated hemichrome formation in ‘hemosomes’ prepared by the ether-injection technique, and increased oxidation of hemoglobin in hand-shaken ‘hemosomes’. The denaturation of hemoglobin in ether-injection ‘hemosomes’ was increased if the initial methemoglobin content of the hemolysate, or the temperature of preparation was elevated. It was slower if liposomes were prepared under either N₂ or CO, or if the radical scavenger 1,3-diphenylisobenzofuran was added with the ether. Egg phosphatidylcholine and synthetic saturated phospholipids gave the same results. With hand-shaken ‘hemosomes’ the oxidized product was primarily methemoglobin, and oxidation could be inhibited by using saturated phosphatidylcholines instead of egg phosphatidylcholine. Lysophosphatidylcholine levels were higher and arachidonic acid levels lower in egg phosphatidylcholine ‘hemosomes’ than in equivalent liposomes containing no hemolysate. The ‘hemosome’ seems to be a suitable model for the study of hemoglobin-lipid membrane interactions and the resulting hemoglobin denaturation process.     

Fluorine-NMR studies of chimpanzee hemoglobin

It has been demonstrated that 4-fluorophenylalanine, a known inhibitor of protein synthesis, becomes incorporated into hemoglobin when present in the diet of a chimpanzee. 19F-NMR spectra of various forms of this protein show well-resolved lines, each line presumably corresponding to a unique phenylalanine/fluorophenylalanine position of the primary sequence. Fluorine chemical shifts and, by implication, tertiary structures vary with the oxidation state and ligand.     

C solution state and solid state n.m.r. of wheat gluten

¹³C solid state and solution state, high resolution n.m.r. spectra have been used to characterize wheat gluten both as dry solid and hydrated mass. Most peaks in the spectra have been satisfactorily assigned. Four different environments within the material have been distinguished corresponding to rigid immobile protein, mobile hydrophobic side chains, mobile lipid and a mobile protein fraction apparent in the hydrated material.     

Saccharose solid matrix embedded proteins: a new method for sample preparation for X-ray absorption spectroscopy

In this study, solid samples of hemoglobin and hemocyanin have been prepared by embedding the proteins into a saccharose-based matrix. These materials have been developed specifically for specimens for X-ray absorption spectroscopy (XAS). The preservation of protein conformation and active site organization was tested, making comparisons between the solid and the corresponding liquid samples, using resonance Raman, infra red, fluorescence and XAS. The XAS spectra of irradiated solid and liquid samples were then compared, and the preservation of biological activity of the proteins during both preparation procedure and X-ray irradiation was assessed. In all cases, the measurements clearly demonstrate that protein solid samples are both structurally and functionally quite well preserved, much better than those in the liquid state. The saccharose matrix provides an excellent protection against X-ray damages, allowing for longer exposure to the X-ray beam. Moreover, the demonstrated long-term stability of samples permits their preparation and storage in optimal conditions, allowing for the repetition of data collection with the same sample in several experimental sessions. The very high protein concentration that can be reached results in a significantly better signal-to-noise ratio, particularly useful for high molecular weight proteins with a low metal-to-protein ratio. On the bases of the above-mentioned results, we propose the new method as a standard procedure for the preparation of biological samples to be used for XAS spectroscopy. Received: 3 August 1999 / Revised version: 5 April 2000 / Accepted: 6 April 2000     

Interactions of phosphatidylinositol 3-kinase Src homology 3 domain with its ligand peptide studied by absorption, circular dichroism, and UV resonance raman spectroscopies

Absorption, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopies were applied to selectively examine the environmental and structural changes of Trp and Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain induced by ligand association. Comparison of the spectra of PI3K SH3 in the presence or absence of its ligand peptide RLP1 (RKLPPRPSK) indicated that RLP1 binding changed the environment of Trp55 of the SH3 to be more hydrophilic and its H bonding weaker and that of Tyr residues to be more hydrophobic. The D21N mutant (Asp21 --> Asn) of the SH3 yielded a UV CD distinct from that of the wild type, and its spectral changes induced by RLP1 binding were smaller and different from those of the wild type in absorption, CD, and UVRR spectra, suggesting that the mutation of conserved Asp21 affected the conformation of the ligand binding cleft and thus might lead to the decrease in the ligand affinity. These data provide direct evidence for the occurrence of environmental and structural changes of PI3K SH3 by the association of a ligand and the D21N mutation.     

Saturation-transfer electron paramagnetic resonance detection of anisotropic motion by sickle hemoglobin molecules in the polymer state

The motional behavior of spin-labeled deoxygenated sickle hemoglobin has been studied by using both 9- and 35-GHz saturation-transfer electron paramagnetic resonance (EPR). Using spectral subtraction techniques and saturation-transfer EPR parameter correlation plots, we find that the saturation-transfer EPR spectra for the sickle hemoglobin gel state at high temperature and high hemoglobin concentration cannot be described as a simple superposition of spectra from immobilized hemoglobin plus solution-state hemoglobin but instead suggest that the individual sickle hemoglobin molecules exhibit limited, anisotropic, rotational oscillation within the polymer fiber. The spectra also imply that the symmetry axis for sickle hemoglobin rotational oscillation is approximately coincident with the nitroxide z axis of the covalently attached spin-label. We suggest that this anisotropic rotational motion may be produced by one or two of the known intermolecular contact sites within the sickle hemoglobin fiber acting as strong intermolecular binding sites, and producing "motional alignment" within the fiber; determining the location of the strong binding site should be important in focusing the future development of antisickling agents.     

Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.     

Application of ATR‐FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state

FTIR spectroscopy in combination with ATR sampling technique is the most accessible analytical technique to study secondary structure of proteins both in solid and aqueous solution. Although several studies have demonstrated the applications of ATR‐FTIR to study conformational changes of solid dried proteins due to dehydration, there are no reports that demonstrate the application of ATR‐FTIR in the study of thermally induced changes of secondary structure of biomolecules directly on the solid state. In this study, four biomolecules of pharmaceutical interest, lysozyme, myoglobine, chymotripsin and human growth hormone (hGH), were studied on the solid state before and after different thermal treatments in order to relate changes of secondary structure to partial or total thermal denaturation processes. The results obtained provide experimental evidence that protein thermal denaturation in the solid state can be detected by displacement of carbonyl bands which correspond to conformational transformations between α–helix to β‐sheet or intermolecular β‐sheet; the molecules studied undergo this transformation when exposed to a temperature close to their denaturation temperature which may become irreversible depending on the extent of the heating treatment. These findings demonstrate that ATR‐FTIR is an effective and time efficient technique that allows the monitoring of the protein thermal denaturation process of solid samples without further reconstitution or prior sample preparation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 574–584, 2015.