Sperm-activating proteins from unfertilized eggs of the Pacific herring, Clupea pallasii

Ripe unfertilized eggs of the Pacific herring, Clupea pallasii , release sperm-activating proteins into seawater at the time of fertilization. Five species of herring sperm-activating proteins (HSAP) with different pl values (4.8, 4.9, 5.0, 5.1 and 5.4) were purified from the egg-conditioned medium by gel filtration and isoelectric focusing. Molecular mass of the HSAP (pl = 5.1), the major species of the five HSAP, was determined to be 8.1 kDa by mass spectrometry. Molecular weights of all of the HSAP were estimated to be 7700 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The first 20 amino acid sequences from N-terminal ends of three HSAP (pl = 4.9, 5.0 and 5.1) were almost identical, suggesting that the HSAP have similar structures.     

Rotational correlation times of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label with respect to heme and nonheme proteins

Noncovalent spin labeled proteins (ovalbumin, bovine serum albumin, hemoglobin, and cytochrome c) were investigated in order to follow the different type of interactions between the nitroxide radical of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label and functional groups of heme and nonheme proteins as well as the pH influence on molecular motion of the label with respect to these proteins. EPR spectra were recorded at room temperature and the computer simulation analysis of spectra was made in order to obtain the magnetic parameters. Noncovalent labeling of proteins can give valuable information on the magnetic interaction between the label molecule and the paramagnetic center of the proteins. The relevance of this interaction can be obtained from line shape analysis: computer simulations for nonheme proteins assume a Gaussian line shape, whereas for heme proteins, a weighted sum of Lorentzian and Gaussian components is assumed. In the framework of the "moderate jump diffusion" model for rotational diffusion, the rotational correlation time is strongly influenced by pH, because of the electrostatic interactions and hydrogen bonding.     

Proton nuclear magnetic resonance characterization of the oxidized intermediates of cytochrome c peroxidase

Oxidation of cytochrome c peroxidase with hydrogen peroxide to form the initial oxidized intermediate, cytochrome c peroxidase compound I, drastically alters the proton hyperfine nmr spectrum. In contrast to studies of horseradish peroxidase, where the spectrum of horseradish peroxidase compound I is similar to that of the native protein, cytochrome c peroxidase compound I exhibits only broad resonances near 17 and 30 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate. No unique resonances attributable to cytochrome c peroxidase compound II could be identified. These results define the molecular conditions for which resolved hyperfine resonances of the iron(IV) states of heme proteins may be observed when the data presented here are compared with the data from horseradish peroxidase. Oxidation of cytochrome c peroxidase while it is complexed to ferricytochrome c reveals that the heme resonances of cytochrome c are not influenced by the oxidation state of cytochrome c peroxidase.     

Structural and dynamic properties of the heme pocket in myoglobin probed by optical spectroscopy

We report the optical absorption spectra of sperm whale deoxy-, oxy-, and carbonmonoxymyoglobin in the temperature range 300–20 K and in 65% glycerol or ethylene glycol–water mixtures. By lowering the temperature, all bands exhibit half-width narrowing and peak frequency shift; moreover, the near-ir bands of deoxymyoglobin show a marked increase of the integrated intensities. Opposed to what has already been reported for human hemoglobin, the temperature dependence of the first moment of the investigated bands does not follow the behavior predicted by the harmonic Franck–Condon approximation and is sizably affected by the solvent composition; this solvent effect is larger in liganded than in nonliganded myoglobin. However, for all the observed bands the behavior of the second moment can be quite well rationalized in terms of the harmonic Franck–Condon approximation and is not dependent on solvent composition. On the basis of these data we put forward some suggestions concerning the structural and dynamic properties of the heme pocket in myoglobin and their dependence upon solvent composition. We also discuss the different behaviors of myoglobin and hemoglobin in terms of the different heme pocket structures and deformabilities of the two proteins.     

Heme conjugated magnetic beads to isolate heme-binding proteins from complex mixtures

The cofactor heme (Fe-protoporphyrin IX) plays many important roles in biology. Identification of novel proteins for the transport, chaperoning and delivery of heme in cells is of widespread interest. Here, we describe the use of heme conjugated magnetic beads for the isolation of heme-binding proteins from complex protein mixtures. The reagent is straightforward to use, sensitive and specific.     

The cytoplasmic heme-binding protein (PhuS) from the heme uptake system of Pseudomonas aeruginosa is an intracellular heme-trafficking protein to the delta-regioselective heme oxygenase

The uptake and utilization of heme as an iron source is a receptor-mediated process in bacterial pathogens and involves a number of proteins required for internalization and degradation of heme. In the following report we provide the first in-depth spectroscopic and functional characterization of a cytoplasmic heme-binding protein PhuS from the opportunistic pathogen Pseudomonas aeruginosa. Spectroscopic characterization of the heme-PhuS complex at neutral pH indicates that the heme is predominantly six-coordinate low spin. However, the resonance Raman spectra and global fit analysis of the UV-visible spectra show that at all pH values between 6 and 10 three distinct species are present to varying degrees. The distribution of the heme across multiple spin states and coordination number highlights the flexibility of the heme environment. We provide further evidence that the cytoplasmic heme-binding proteins, contrary to previous reports, are not heme oxygenases. The degradation of the heme-PhuS complex in the presence of a reducing agent is a result of H2O2 formed by direct reduction of molecular oxygen and does not yield biliverdin. In contrast, the heme-PhuS complex is an intracellular heme trafficking protein that specifically transfers heme to the previously characterized iron-regulated heme oxygenase pa-HO. Surface plasmon resonance experiments confirm that the transfer of heme is driven by a specific protein-protein interaction. This data taken together with the spectroscopic characterization is consistent with a protein that functions to shuttle heme within the cell.     

高脂血症性急性重症胰腺炎特征分析及治疗策略

目的:探讨高脂血症性急性重症胰腺炎(HSAP)患者的临床特点及诊疗分析,为急性重症胰腺炎(SAP)的诊治提供偱证依据。方法:回顾性分析了我科自2008年9月到2013年9月收治的101例急性胰腺炎患者血清脂肪酶、淀粉酶浓度、酶恢复时间、白细胞、甘油三酯差异以分析高脂血症性急性重症胰腺炎患者的病情转归情况。结果:HSAP患者甘油三酯(TG)水平男性明显高于女性,差异有统计学意义(P0.05);HSAP患者入院后经过降脂药物治疗HSAP与其他SAP相比较甘油三酯(TG)在短期内基本恢复正常,HSAP中白细胞、酶恢复时间低于其他SAP组,差异有统计学意义(P0.05);HSAP与高脂血症性急性轻型胰腺炎(HMAP)相比较两组间淀粉酶、脂肪酶、甘油三酯比较差异无统计学意义(P0.05),白细胞、酶恢复时间比较差异有统计学意义(P0.05)。结论:HSAP患者TG水平可能与患者的性别有一定的相关性;HSAP患者的病情转归可能与甘油三酯水平相关;甘油三酯水平不能直接决定高脂血症性急性胰腺炎患者病情的轻重。     

Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixture

The structure of horseradish peroxidase (HRP) in phosphate buffered saline (PBS)/dimethyl sulfoxide (DMSO) mixed solvents at different compositions is investigated by IR, electronic absorption, and fluorescence spectroscopies. The fluorescence spectra and the amide I spectra of ferric HRP [HRP(Fe3+)] show that overall structural changes are relatively small up to 60% DMSO. Although the amide I band of HRP(Fe3+) shows a gradual change in the secondary structure and a decrease in the contents of a helices, its fluorescence spectra indicate that the distance between the heme and Trp173 is almost constant. In contrast, the changes in the positions of the Soret bands for resting HRP(Fe3+) and catalytic intermediates (compounds I and II) and the IR spectra at the C-O stretching vibration mode of carbonyl ferrous HRP [HRP(Fe2+)-CO] show that the microenvironment in the distal heme pocket is altered, even with low DMSO contents. The large reduction of the catalytic activity of HRP even at low DMSO contents can be attributed to the structural transition in the distal heme pocket. In PBS/DMSO mixtures containing more than 70 vol % DMSO, HRP undergoes large structural changes, including a large loss of the secondary structure and a dissociation of the heme from the apoprotein. The presence of the components of the amide I band that can be assigned to strongly hydrogen bonding amide C=O groups at 1616 and 1684 cm(-1) suggests that the denatured HRP may aggregate through strong hydrogen bonds.     

Resonance Raman spectra of bovine liver catalase: enhancement of proximal tyrosinate vibrations

Resonance Raman spectra of native bovine liver ferri-catalase have been obtained in the 200-1800 cm-1 region. Excitation at a series of wavelengths ranging from 406.7 to 514.5 nm has been used and gives rise to distinct sets of resonance Raman bands. Excitation within the Soret and Q-bands of the heme group produces the expected set of polarized and nonpolarized porphyrin modes, respectively. The frequencies of the porphyrin skeletal stretching bands in the 1450-1700 cm-1 region indicate that catalase contains only five-coordinate, high-spin heme groups. In addition to the porphyrin modes, bovine liver catalase exhibits bands near 1612 and 1520 cm-1 that are attributable to ring vibrations of the proximal tyrosinate that are enhanced via resonance with a proximal tyrosinate----Fe(III) change transfer transition centered near 490 nm. Similar bands have been observed in mutant hemoglobins that have tyrosinate axial ligands and in other Fe(III)-tyrosinate proteins. No resonance Raman bands have been observed that can be attributed to degraded hemes. The spectra are relatively insensitive to pH over the range of 5-10, and the same spectra are observed for catalase samples that do and do not contain tightly bound NADPH. Resonance Raman spectra of the fluoride complex exhibit porphyrin skeletal stretching modes that show it to be six coordinate, high spin, while the cyanide complex is six coordinate, low spin. Both the azide and thiocyanate complexes, however, are spin-state mixtures with the high-spin form predominant.     

Detection of proteins and phenol in DNA samples with second-derivative absorption spectroscopy.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.     

Negative ion chemical ionization mass spectrometry of 2,4-dinitrophenyl amino acid esters

We report the negative ion chemical ionization mass spectra of 2,4-dinitrophenyl (DNP) amino acid methyl esters. For the common amino acids, these derivatives exhibit very simple mass spectra; the molecular anion is the base peak in all cases. The electrophilicity of the DNP group allows selective and sensitive ionization. Amino acids can be identified singly or in mixtures by their molecular weights, and picomole detection is possible. Chromatography is only needed for Leu/Ile differentiation. Amino-terminal analysis of proteins is demonstrated.     

Spectroscopic characterization of (57)Fe-enriched cytochrome c

Investigation of the heme iron dynamics in cytochrome c with Mössbauer spectroscopy and especially nuclear resonance vibrational spectroscopy requires the replacement of the natural abundant heme iron with the ⁵⁷Fe isotope. For demetallization, we use a safer and milder ferrous sulfate–hydrochloric acid method in addition to the harsher commonly used hydrofluoric acid-based procedure. The structural integrity of the ⁵⁷Fe-reconstituted protein in both oxidation states is confirmed from absorption spectra and a detailed analysis of the rich resonance Raman spectra. These results reinforce the application of metal-substituted heme c proteins as reliable models for the native proteins.     

Detection of heme oxygenase activity in a library of four-helix bundle proteins: towards the de novo synthesis of functional heme proteins

Design and chemical synthesis of de novo heme proteins with enzymatic activity on cellulose membranes is described. 352 antiparallel four-helix bundle proteins with a single histidine for heme ligation were assembled from three different sets of short amphipathic helices on membrane-bound peptide templates. The templates were coupled by linkers to cellulose membranes of microplate format, which could be cleaved for control of intermediate and final products. The incorporation of heme and the heme oxygenase activity of the 352 proteins were monitored by measuring UV-visible spectra directly on the cellulose. The kinetics of the heme oxygenase reaction was studied by monitoring the decrease of the Soret band and the transient absorbance of verdoheme being an intermediate product in the formation of biliverdin. Four of the proteins covering a broad range of the enzymatic rate constants were selected and synthesized in solution for further characterization. Detailed studies by redox potentiometry, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy yielded information about the aggregation state of the proteins, the spin state and the putative coordination environment of the iron. The amount of five-coordinated high-spin iron and a positive reduction potential were found to promote the oxygenase activity of the proteins.     

The quantum mixed-spin heme state of barley peroxidase: A paradigm for class III peroxidases.

Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.     

细胞色素C与细胞色素氧化酶之间相互作用的共振RAMAN光谱研究

分别于５１４．５ｎｍ及６０４ｕｍ波长激发下,对游离的细胞色素Ｃ,细胞色素氧化酶以及细胞色素Ｃ和细胞色素氧化酶的复合体的共振拉曼光谱进行了分析比较,在形成复合体时,双方蛋白的共振拉曼谱均有所变化,一个共同的特征性变化是Ａ２ｇｖ２２１１３０ｃｍ－１,ｖ２１１３１２ｃｍ－１,ｖ２０１４００ｃｍ－２,和ｖ１９１５８４ｃｍ－１强度都有增强,其中变化最明显的是Ａ２ｇｖ１９１５８４ｃｍ－１峰,在游离态中,Ｉ１５４０／ｉ１５８２＞１,在结合态中Ｉ１５５０／Ｉ１５８２＜１。     

Structure and ligand selection of hemoglobin II from Lucina pectinata

Lucina pectinata ctenidia harbor three heme proteins: sulfide-reactive hemoglobin I (HbI(Lp)) and the oxygen transporting hemoglobins II and III (HbII(Lp) and HbIII(Lp)) that remain unaffected by the presence of H(2)S. The mechanisms used by these three proteins for their function, including ligand control, remain unknown. The crystal structure of oxygen-bound HbII(Lp) shows a dimeric oxyHbII(Lp) where oxygen is tightly anchored to the heme through hydrogen bonds with Tyr(30)(B10) and Gln(65)(E7). The heme group is buried farther within HbII(Lp) than in HbI(Lp). The proximal His(97)(F8) is hydrogen bonded to a water molecule, which interacts electrostatically with a propionate group, resulting in a Fe-His vibration at 211 cm(-1). The combined effects of the HbII(Lp) small heme pocket, the hydrogen bonding network, the His(97) trans-effect, and the orientation of the oxygen molecule confer stability to the oxy-HbII(Lp) complex. Oxidation of HbI(Lp) Phe(B10) --> Tyr and HbII(Lp) only occurs when the pH is decreased from pH 7.5 to 5.0. Structural and resonance Raman spectroscopy studies suggest that HbII(Lp) oxygen binding and transport to the host bacteria may be regulated by the dynamic displacements of the Gln(65)(E7) and Tyr(30)(B10) pair toward the heme to protect it from changes in the heme oxidation state from Fe(II) to Fe(III).     

Experimental and Theoretical Protein Folding

Abstract

Resonance Raman spectra with Q-band excitation are reported for microperoxidase-11, the cytochrome c analog. Spectra were acquired in the mid-frequency range for the oxidized, and reduced forms of the undecapeptide, as well as for the imidazole and carbonyl complexes. Oxidation and spin state marker bands of the undecapeptides are consistent with a six-coordinate, low spin iron in both oxidation states. Porphyrin core size correlations yield a porphyrin-centre to pyrrole-nitrogen distance of 2.00 Å for MP11, suggestive of a six-coordinate species in a distorted heme environment. Molecular dynamics results show that the non-planarity of the heme of the parent cytochrome is conserved in the microperoxidase and its carbonmonoxy analog.     

1H NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A

The reaction of heme and apoprotein has been studied in detail in 1H NMR spectroscopy in order to elucidate the conditions for reconstitution of hemoglobin (Hb) to yield the native protein. The initially formed holoprotein exists as a mixture of isomers with individual subunits possessing the two heme orientations differing by a 180 degrees rotation about the alpha, gamma-meso axis [La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., & Pandey, R. K. (1985) Biochemistry 24, 3826-3831]. We characterize in detail herein the rates and mechanism of heme reorientation and show that the rates differ dramatically for met-aquo and met-azido derivatives and are highly pH dependent in both subunits in a fashion that allows selective equilibration in either subunit. Nonequilibrium mixtures of such isomers can be kinetically trapped in the met-azido form and stored in this metastable form for many months. With kinetically controlled heme orientationally disordered Hb, unambiguous assignment of 1H NMR resonances to individual subunits has been made for the met-azido derivative, which demonstrates approximately 2% and 10% equilibrium heme disorder in the alpha- and beta-subunits, respectively. Comparison of the 1H NMR spectra of various heme rotationally disordered Hb derivatives indicates that this disorder is observable in all forms studied, but is most easily recognized as heme disorder and most conveniently monitored in the met-azido complex. Structural consequences of heme disorder appear to manifest themselves much more strongly in peripheral than axial interactions at the heme. Preliminary studies reveal that both the rate of autoxidation of oxy-Hb and the azide affinity of met-aquo-Hb depend on the orientation of the heme.     

An Analysis of the Electron Spin Resonance of Low Spin Ferric Heme Compounds

The electron spin resonance spectra of low spin ferric heme proteins are calculated and compared with experimental spectra recently obtained for a large number of heme proteins. On the basis of observed g values, these heme proteins have been categorized into five prototypes. Since electron spin resonance spectra are quite sensitive to small changes in the local environment about the paramagnetic ion, we have explored in some detail, the possibility that the g value variation in these different types of proteins is due to systematic environmental changes. To this end, we have calculated the g values as a function of tetragonal and rhombic distortions and compared our results with observed behavior. In addition we have also calculated the energy intervals between the low spin states in zero magnetic field and the temperature dependent magnetic moments and studied the effect on these properties also of small symmetry changes.