Ligand binding intermediates of nitrosylated human hemoglobin induced at low temperature by X-ray irradiation

Under prolonged X-ray irradiation, the ferrous heme of nitrosylated human adult hemoglobin derivative (HbNO) undergoes a reversible transition generating a 5-coordinate species, due to release of the Fe-NO bond. The overall process can be investigated using X-ray absorption near edge structure (XANES) spectroscopy. In this work, Fe K-edge XANES spectra were measured at T < 15 K, pH 9.2, i.e., on a high-affinity state (R-HbNO) where all the hemes are 6-coordinate, and at pH 6.5 in the presence of inositol hexakis-phosphate (IHP), i.e., on a low-affinity ligated state (T-HbNO) where the iron-hemes of the α-chains are 5-coordinate due to breaking of the Fe-proximal histidine bond. Under X-ray irradiation, 5-coordinate Fe-hemes are populated in both R-HbNO and T-HbNO, the Fe-NO bond lysis induced in T-HbNO involving rebinding of the proximal histidine to the transiently populated 4-coordinate hemes of the α-chains. A detailed analysis of the spectra confirms that different intermediate states in the ligand binding cooperative process of hemoglobin can be populated by X-ray irradiation, and that the part of the energy associated to the R-T quaternary transition, that is transmitted to the heme site, can be monitored by XANES spectroscopy.     

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

The native structure of hemoglobin (Hb) comprises two alpha- and two beta-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded alpha- and beta-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric alpha- and beta-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded alpha- and beta-globin. However, some unexpected features are observed, e.g., a heme-deficient beta-globin dimer that represents an off-pathway intermediate. Monomeric beta-globin is capable of binding heme before forming a complex with an alpha-subunit. This observation suggests that holo-alpha-apo-beta globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes.     

Observation of symmetric denaturation of hemoglobin subunits by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been used to characterize the denaturation of porcine hemoglobin (Hb) induced by solvent changes. This work provides evidence for the symmetric nature of Hb denaturation and demonstrates that heme losses from α- and β-monomers occur in parallel, in response to the addition of acid and organic co-solvents in solution. When subject to one of the following solution conditions (pH 3.2-4.0 or 15-30% acetonitrile-water or 30-45% methanol-water solution), α- and β-globins undergo symmetric dissociation to release the heme groups, which is detected by ESI-MS. Circular dichroism (CD) and fluorescence spectroscopy (FS) data show that the acid-induced and organic solvent-induced heme release, as observed in the mass spectra, can probably be ascribed to different aspects of the conformational changes taking place in the protein. The acidity of the solvent has a significant effect on the secondary structure, whereas organic content level in solution (15-30% acetonitrile or 30-45% methanol) tends to destroy the tertiary structure of Hb globins, both leading to release of the heme from each subunit.     

Small‐Angle Neutron Scattering Studies of Hemoglobin Confined Inside Silica Tubes of Varying Sizes

In addition to the chemical nature of the surface, the dimensions of the confining host exert a significant influence on confined protein structures; this results in immense biological implications, especially those concerning the enzymatic activities of the protein. This study probes the structure of hemoglobin (Hb), a model protein, confined inside silica tubes with pore diameters that vary by one order of magnitude (≈20–200 nm). The effect of confinement on the protein structure is probed by comparison with the structure of the protein in solution. Small‐angle neutron scattering (SANS), which provides information on protein tertiary and quaternary structures, is employed to study the influence of the tube pore diameter on the structure and configuration of the confined protein in detail. Confinement significantly influences the structural stability of Hb and the structure depends on the Si‐tube pore diameter. The high radius of gyration (R_g) and polydispersity of Hb in the 20 nm diameter Si‐tube indicates that Hb undergoes a significant amount of aggregation. However, for Si‐tube diameters greater or equal to 100 nm, the R_g of Hb is found to be in very close proximity to that obtained from the protein data bank (PDB) reported structure (R_g of native Hb=23.8 Å). This strongly indicates that the protein has a preference for the more native‐like non‐aggregated state if confined inside tubes of diameter greater or equal to 100 nm. Further insight into the Hb structure is obtained from the distance distribution function, p(r), and ab initio models calculated from the SANS patterns. These also suggest that the Si‐tube size is a key parameter for protein stability and structure.     

Spectroscopic studies on the interaction between human hemoglobin and CdS quantum dots

The interaction between human adult hemoglobin (Hb) and bare CdS quantum dots (QDs) was investigated by fluorescence, synchronous fluorescence, circular dichroism (CD), and Raman spectroscopic techniques under physiological pH 7.43. The intrinsic fluorescence of Hb is statically quenched by CdS QDs. The quenching obeys the Stern-Volmer equation, with an order of magnitude of binding constant (K) of 10(7). The electrostatic adsorption of Hb on the cationic CdS QDs surface is energetically favorable (DeltaS(0)=70.22 Jmol(-1)K(-1), DeltaH(0)=-23.11 kJmol(-1)). The red shift of synchronous fluorescence spectra revealed that the microenvironments of tryptophan and tyrosine residues at the alpha(1)beta(2) interface of Hb are disturbed by CdS QDs, which are induced from hydrophobic cavities to a more exposed or hydrophilic surrounding. The secondary structure of the adsorbed Hb has a loose or extended conformation for which the content of alpha-helix has decreased from 72.5 to 60.8%. Moreover, Raman spectra results indicated that the sulfur atoms of the cysteine residues form direct chemical bonds on the surface of the CdS QDs. The binding does not significantly affect the spin state of the heme iron, and deoxidation is not expected to take place on the coated oxyhemoglobin. The change of orientation of heme vinyl groups was also detected.     

Mobility of spin probes having a primary,secondary, tertiary,or quaternary amino group in drawn and undrawn nylon 6 film

The mobility of spin probes having a secondary, tertiary, or quaternary amino group in dried nylon films was investigated by means of electron spin resonance (ESR) measurements and compared with the behavior of previously investigated spin probes having a primary amino group, a carboxylate group, or a sulfate group. The spin probes having a primary or secondary amino group showed effects of drawing on the mobility, while the other probe molecules did not. This result could be interpreted by considering the interactions between the spin probes and the nylon chains. In the undrawn nylon film, the mobilities of the nonionic spin probes were almost the same, and smaller than those of the charged spin probes, suggesting that the location in the nylon film is different for the uncharged and charged spin probes. These results are discussed in detail using separation of extrema of the ESR spectra, rotational correlation times, and anisotropy parameters.     

光谱法研究尿素对水溶液中血红蛋白构象的影响

应用荧光猝灭法和动态光散射法测定尿素-水混合溶剂中血红蛋白(Hb)与联苯胺的结合距离和Hb的流体动力学半径. 结合Hb的荧光光谱和吸收光谱, 探讨尿素与蛋白质分子在水溶液中相互作用的机理及其对蛋白质构象的影响. 结果显示, 尿素分子取代水分子在蛋白质周围形成溶剂化层, 并与骨架肽链和亲水侧链形成氢键, 从而积聚在蛋白质分子表面. 尿素分子与蛋白质分子之间的直接相互作用对蛋白质的构象具有复杂的影响, 高浓度的尿素-水混合溶剂破坏蛋白质的构象, 而低浓度的混合溶剂则有利于蛋白质形成更紧密的构象. 在高浓度的尿素-水混合溶剂中, Hb血红素疏水空穴失去原有的三级结构后形成一个与熔球态相类似的结构.     

Effects of dynamical couplings in IR spectra of the hydrogen bond in N-phenylacrylamide crystals

This article presents the investigation results of the polarized IR spectra of the hydrogen bond in N-phenylacrylamide crystals measured in the frequency range of the proton and deuteron, ν(N-H) and ν(N-D), stretching vibration bands. The basic spectral properties of the crystals were interpreted quantitatively in terms of the "strong-coupling" theory. The proposed model of the centrosymmetric dimer of hydrogen bonds facilitated the explanation of the well-developed, two-branch structure of the ν(N-H) and ν(N-D) bands as well as the isotopic dilution effects in the spectra. The vibronic mechanism of the generation of the long-wave branch of the ν(N-H) band ascribed to the excitation of the totally symmetric proton vibration was elucidated. The complex fine structure pattern of ν(N-H) and ν(N-D) bands in N-phenylacrylamide spectra in comparison with the spectra of other secondary amide crystals (e.g., N-methylacetamide and acetanilide) can be accounted for in terms of the vibronic model for the forbidden transition breaking in the dimers. On the basis of the linear dichroic and temperature effects in the polarized IR spectra of N-phenylacrylamide crystals, the H/D isotopic "self-organization" effects were revealed.     

Proton nuclear magnetic resonance and electron paramagnetic resonance investigation of alpha-alpha cross-linked Fe-Co hybrid hemoglobins.

The following asymmetric alpha 1 99 Lys-alpha 2 99 Lys cross-linked Fe(II)-Co(II) hybrid hemoglobins (Hbs) were first prepared from derivatives of hemoglobin C (beta 6 Glu-Lys) and human normal HbA: [alpha(Co)beta(Fe)]A[alpha(Co)beta(Co)]cXL, [alpha(Fe)beta(Co)]A[alpha(Co)beta(Co)]cXL, etc. Their 500 MHz 1H NMR and EPR spectra were measured in order to study the change in their tertiary and quaternary structure under atmosphere of deoxy, oxy and carbon monoxide (with or without IHP). From the change of T and R marks in 1H NMR hydrogen bonding region, it is proved that oxygen molecules are first bonded to alpha(Fe) subunits rather than to beta(Fe). The experimental phenomena provided further evidence that intermediate states of ligation are present in addition to T and R state during process of binding of oxygen to Hb. IHP facilitates transformation of T state to R state. The same conclusion can also be drawn from the results of EPR spectra at 77 K.     

Infrared and microwave spectroscopy of simple hydrogen-bonded dimers in gas-phase systems

The structures of ν_S-bands in infrared spectra and of rotational spectra in the microwave region, for simple dimers B?H-A in the gas phase, are reviewed. In infrared spectra an overall sub-band structure arises from a progression in the hydrogen bond stretching mode ν_σ, while each of these sub-bands can have structure arising from a hot-band sequence in the hydrogen-bond bending mode ν_β, and these sequence bands can have rotational fine structure forming a P-branch band-head. In microwave spectra a progression of satellites based on ν_β generally dominates the spectrum, although a weaker similar progression in ν_β based on the state ν_σ = 1 can also occur.     

新型季铵型阳离子表面活性剂的合成及性能

以环氧大豆油为基本原料合成了一种季铵型阳离子表面活性剂,通过红外光谱和核磁共振氢谱对其结构进行了表征,并对其表面张力和临界胶束浓度进行了测定.     

Direct electrochemistry of hemoglobin in layer-by-layer films with poly(vinyl sulfonate) grown on pyrolytic graphite electrodes

Stable layer-by-layer electroactive films were grown on pyrolytic graphite (PG) electrodes by alternate adsorption of layers of polyanionic poly(vinyl sulfonate) (PVS) and positively charged hemoglobin (Hb) from their aqueous solutions. Cyclic voltammetry (CV) of [PVS/Hb]n films showed a pair of well-defined and nearly reversible peaks at about - 0.28 V vs. SCE at pH 5.5, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The process of (PVS/Hb) bilayer growth was monitored and confirmed by CV, X-ray photoelectron spectroscopy (XPS) and UV-Vis spectroscopy. While the amount of Hb adsorbed in each bilayer was the same, the amount of electroactive Hb in each bilayer decreased dramatically with increase of the number of bilayer, and electroactivity was just extended to 8 [PVS/Hb] bilayers. CVs of [PVS/Hb]8 films maintained stable in buffers containing no Hb. Positions of Soret band of Hb in [PVS/Hb]n films grown on transparent glass slides suggest that Hb in the films keeps its secondary structure similar to its native state in a wide pH range. Trichloroacetic acid and nitrite were catalytically reduced by [PVS/Hb]8 films with significant lowering of the electrode potential required.     

Pseudomonas cytochrome c peroxidase. XIII. pH-denaturation of the enzyme.

The effect of pH on the oxidized Pseudomonas cytochrome c peroxidase molecule was studied by measuring the peroxidatic activity, the sedimentation velocity, the circular dichroic spectra in the far UV and Soret regions, and the optical absorption spectra of the enzyme in the pH range 2.5-13.0 at a constant ionic strength (micron = 0.1). The enzyme was stable in a narrow pH region, pH 6.0 - 7.4. In the low pH range the gross tertiary structure was observed to change quite simultaneously with the enzymatic activity and secondary structure. The optical absorption spectra indicated that there were no coordinated internal protein liqands in the 6th coordination positions of the heme prosthetic groups at the lowest pH studied. In the high pH range the secondary structure and the protein environment of hemes were observed to remain stable after the tertiary structure had changed and the activity had decreased. According to the optical absorption spectra the 6th internal protein ligands of hemes were retained at the highest pH studied.     

The Photostabilization of Hemoglobin in Triton X-100 Aqueous Solution and Triton X-100/n-C5H11OH/H2O Microemulsion

The influence of UV-irradiation on the interaction of hemoglobin (Hb) with Triton X-100 is investigated by UV–Vis absorption spectroscopy, fluorescence spectroscopy and freeze-fractured transmission electron microscopy. It is found that in Triton X-100/H₂O systems Hb can convert to hemichrome but heme is not present, whereas in Triton X-100/n-C₅H₁₁OH/H₂O microemulsion Hb can convert to hemichrome and then induce the heme monomer to leave the hydrophobic cavity of Hb. UV-irradiation can also convert Hb to hemichrome, and subsequently make heme to be photodegraded, in which the conversion rate depends on the structure of the surfactant aggregates. Furthermore, in order to understand the mechanism of photostabilization of Hb in Triton X-100 systems, the photostabilization of heme in the Triton X-100 aqueous solutions and Triton X-100/n-C₅H₁₁OH/H₂O microemulsions has been studied.     

Electrochemically Monitoring the Acid and Acidic Urea‐Induced Unfolding of Hemoglobin and Its Electrocatalytic Ability

Acid and acidic urea‐induced unfolding of hemoglobin (Hb) was investigated using protein‐film‐based electrochemical method. The conformational transition of Hb was monitored through the change of direct electrochemical response of Hb. The heme groups in Hb detached from their native binding sites after pH<4.0. Spectral experiments fully supported the results. Furthermore, the catalytic ability of Hb was enhanced 15 times under the optimal unfolded conditions of pH 2.0 PBS containing 3.0 mol L^?1 urea. The method contributes to understand the relationship between function and conformation of Hb, and provides possibility of manipulating protein function by controlling its conformation.     

In situ fluorescence probing of the chemical and structural changes during formation of hexagonal phase cetyltrimethylammonium bromide and lamellar phase CTAB/Poly(dodecylmethacrylate) sol–gel silica thin films

Surfactant-templated mesostructured sol–gel films formed by evaporation induced self assembly (EISA) exhibit highly-ordered hexagonal, lamellar, and cubic structures. The steady-state dip-coating configuration allows both the chemistry and the dynamics of the EISA process to be traced in real time because the steps involved in the formation of the mesostructured material are separated both spatially and temporally in the dip-coating direction. The dynamic processes occurring during film formation can be conveniently monitored by the combination of interferometry and fluorescence spectroscopy of incorporated molecular probes. The selected probes respond to changes in their rotational mobility and the surrounding solvent composition and report these changes through their fluorescence characteristics. By taking in situ fluorescence spectra at various positions within the progressively thinning film, changes in the solvent composition, onset of micelle formation and further organization to the final mesophase structure can be followed. The luminescence of the probe molecule is measured with a spatial resolution of 100 μm. Two categories of surfactant-templated mesostructured sol–gel films were examined. Cetyltrimethylammonium bromide (CTAB) systems assemble into a 2-D hexagonal surfactant/silica mesophase with the surfactant concentration used in this study. CTAB dodecylmethacrylate systems assemble into a lamellar mesophase, which can be further polymerized to form a poly(dodecylmethacrylate)/silica hybrid nanocomposite that mimics nacre. X-ray diffraction patterns, transmission electron microscopy images, and other techniques are used to characterize the final films.     

Über bismut-haltige heterocyclen: I. Methylierte und phenylierte bismocane,intra- und intermolekulare koordinationserhöhung an bismut(III)

Methyl and phenyl oxadithia and trithiabismocanes have been synthesized from methyl or phenyl diethoxybismutane and the respective dithiol. The light-sensitive compounds have been investigated by mass, vibrational and ¹³C NMR spectra: ν(BiMe) 470–460, ν(BiS₂) 300–240 cm^?1; δ(¹³Me) ?12 ppm. The crystal structure of 5-phenyl-1,4,6,5-oxadithiabismocane has been determined (R = 0.056). The eight-membered ring has the chair-chair conformation. Besides three direct bonds (BiPh 225(2), BiS 256.0(2) and 260.2(3) pm) there are one transannular (Bi?O 297(1) and two intermolecular contacts (Bi ?S 344.0(3) and 350.9(3) pm) to bismuth in resulting a ψ-monocapped octahedral sphere of coordination. These polyhedra are connected in sharing two different edges, and the crystal structure exhibits double chains of molecules.     

十二烷基磺酸钠对大豆过氧化物酶活性和构象的影响

在不同的pH值体系中, 利用酶活测量、圆二色谱、荧光谱和电子吸收谱研究了十二烷基磺酸钠(SDS)对大豆过氧化物酶(SBP)活性与构象的影响情况. 在pH 2.6和4.2 的体系中, 少量的SDS分子可通过静电作用与SBP结合, 进而与SBP分子中的His169残基结合, 降低其与铁卟啉的配位能力, 使其Soret吸收带蓝移, 二级结构发生轻微的变动, 活性永久丧失. 在pH 5.2体系中, SDS和SBP分子都带负电, 由于静电排斥作用, SDS无法进入SBP的分子内部, 失去与SBP分子中His169残基结合的能力, 对SBP分子的二级结构没有影响, 仅对SBP分子的三级结构有所影响. 当SDS的浓度大于临界胶束浓度时, 由于胶束与SBP的静电排斥作用增强, 限制了铁卟啉中乙烯基的运动, 乙烯基与卟啉环的共轭程度增大, Soret 吸收带红移. 由于SBP活性可完全恢复, 此变化是可逆的.     

碳糊电极上无机膜固载血红蛋白的直接电化学

报道了用硅溶胶-凝胶(Sol-gel)膜将血红蛋白(Hb)固载于碳糊电极上的直接电化学行为.研究结果表明,Hb-Sol-gel修饰的碳糊电极在pH=7.0的缓冲溶液中于-0.275V(vs.Ag/AgCl)处有一对可逆的循环伏安氧化-还原峰,为Hb血红素辅基Fe(Ⅲ)/Fe(Ⅱ)电对的特征峰.HbFe(Ⅲ)/Fe(Ⅱ)电对的式量电位在pH5.0～11.0范围内与溶液pH值呈线性关系,表明Hb的电化学还原很可能是一个质子伴随着一个电子的电极过程.FTIR光谱证实,Sol-gel膜对Hb的固载没有破坏其天然结构.Hb-Sol-gel修饰的碳糊电极能够催化还原H₂O₂,可望将其用于制作第三代生物传感器.