Circular dichroism and magnetic circular dichroism of nitrogenase proteins

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from Azotobacter vinelandii (Av) and Klebsiella pneumoniae (Kp) have been obtained in the near infrared-visible-near ultraviolet spectral region. Previously, visible CD was reported to be absent or barely detectable in nitrogenase proteins; MCD spectra have not been reported. The chiroptical spectra can be measured in solution at room temperature, an advantage relative to spectroscopic methods requiring cryogenic sample temperatures. Absorption spectra were also obtained. The CD and MCD are markedly more structured, and thus interpretively more useful, than the corresponding absorption spectra. The dithionite-reduced MoFe proteins (Av1, Kp1) have nearly identical CD and MCD, demonstrating identical numbers and types of metal centers in similar protein environments. The CD and MCD cannot be explained solely in terms of contributions from known 4-Fe or 2-Fe clusters; the near-infrared MCD is inconsistent with the presence of known 4-Fe clusters. CD and MCD spectra of Lauth's violet-oxidized Kp1 are also reported. The reduced Fe proteins (Av2, Kp2) have similar CD and MCD, again indicating significant conservation of chromophore environment. The spectra clearly demonstrate the presence of a reduced bacterial ferredoxin-like (C(3-)) 4-Fe cluster. No obvious evidence of additional chromophores is observed. CD, MCD, and absorption spectra of Av1-oxidized Av2 are reported. The absorption spectrum shows the expected shoulder near 390 nm. The CD and MCD are characteristic of a C(2-) 4-Fe cluster; in particular, the diagnostic near-infrared MCD peak is observed at approximately 8300 cm(-1). The CD of Av2 oxidized in the presence and absence of MgATP are radically different, providing the first direct evidence for MgATP interaction with Fe protein in this oxidation state.     

Bright circularly polarized soft X-ray high harmonics for X-ray magnetic circular dichroism

We demonstrate, to our knowledge, the first bright circularly polarized high-harmonic beams in the soft X-ray region of the electromagnetic spectrum, and use them to implement X-ray magnetic circular dichroism measurements in a tabletop-scale setup. Using counterrotating circularly polarized laser fields at 1.3 and 0.79 µm, we generate circularly polarized harmonics with photon energies exceeding 160 eV. The harmonic spectra emerge as a sequence of closely spaced pairs of left and right circularly polarized peaks, with energies determined by conservation of energy and spin angular momentum. We explain the single-atom and macroscopic physics by identifying the dominant electron quantum trajectories and optimal phase-matching conditions. The first advanced phase-matched propagation simulations for circularly polarized harmonics reveal the influence of the finite phase-matching temporal window on the spectrum, as well as the unique polarization-shaped attosecond pulse train. Finally, we use, to our knowledge, the first tabletop X-ray magnetic circular dichroism measurements at the N_4,5 absorption edges of Gd to validate the high degree of circularity, brightness, and stability of this light source. These results demonstrate the feasibility of manipulating the polarization, spectrum, and temporal shape of high harmonics in the soft X-ray region by manipulating the driving laser waveform.High-harmonic generation (HHG) results from an extreme nonlinear quantum response of atoms to intense laser fields. When implemented in a phase-matched geometry, bright, coherent HHG beams can extend to photon energies beyond 1.6 keV (1, 2). For many years, however, bright HHG was limited to linear polarization, precluding many applications in probing and characterizing magnetic materials and nanostructures, as well as chiral phenomena in general. Although X-ray optics can in principle be used to convert extreme UV (EUV) and X-ray light from linear to circular polarization, in practice such optics are challenging to fabricate and have poor throughput and limited bandwidth (3). A more appealing option is the direct generation of elliptically polarized (4–6) and circularly polarized (7–9) high harmonics. In recent work we showed that by using a combination of 0.8 and 0.4 µm counterrotating driving fields, bright (i.e., phase-matched) EUV HHG with circular polarization can be generated at wavelengths λ > 18 nm and used for EUV magnetic dichroism measurements (10–13).Here we make, to our knowledge, the first experimental demonstration of circularly polarized harmonics in the soft X-ray region to wavelengths λ < 8 nm, and use them to implement soft X-ray magnetic circular dichroism (XMCD) measurements using a tabletop-scale setup. By using counterrotating driving lasers at 0.79 µm (1.57 eV) and 1.3 µm (0.95 eV), we generate bright circularly polarized soft X-ray HHG beams with photon energies greater than 160 eV (14) and with flux comparable to the HHG flux obtained using linearly polarized 800-nm driving lasers (15). Moreover we implement, to our knowledge, the first advanced simulations of the coherent buildup of circularly polarized high harmonics to show how the macroscopic phase-matching physics and ellipticity of the driving lasers influence the HHG spectra, number of bright attosecond bursts, and the degree of circular polarization.This work presents several new capabilities and findings. First, circularly polarized HHG provides a unique route for generating bright narrowband (λ/Δλ > 400) harmonic peaks in the soft X-ray region, to complement the soft X-ray supercontinua that are produced with linearly polarized mid-IR lasers (2, 15, 16). This capability is significant because it provides an elegant and efficient route for shaping soft X-ray light by manipulating the driving laser light, and is very useful for applications in high-resolution coherent imaging (17–21) and photoelectron spectroscopies. Second, we show that the macroscopic phase-matching physics of circularly polarized soft X-ray HHG driven by mid-IR lasers has similarities to linearly polarized HHG, where the number of bright attosecond bursts is limited by the finite phase-matching temporal window. Third, we implement the first tabletop XMCD measurements at the N_4,5 absorption edges of Gd. The Gd/Fe multilayer sample is a candidate material for next-generation all-optical magnetic storage devices (22), but has been inaccessible to HHG XMCD until now. This capability also opens up the possibility of probing spin dynamics in rare-earth elements using HHG, which has been successfully used for 3d transition metals to uncover the fastest spin dynamics using EUV HHG (23, 24). Finally, and most importantly, these results demonstrate the universal nature of circularly polarized HHG that can be generated across the EUV and soft X-ray spectral regions using a broad range of driving laser wavelengths.     

Magnetic circular dichroism of peralkylated tetrasilane conformers

Magnetic circular dichroism (MCD) of five peralkylated tetrasilanes (1-5) conformationally constrained to angles ranging from nearly 0 degrees to 180 degrees and of the open chain tetrasilane Si(4)Me(10) (6) shows a clear conformational dependence and permits the detection of previously hidden transitions. In the tetrasilane CH(2)Si(4)Me(8) (1), with the smallest dihedral angle, comparison of MCD with absorption spectra reveals four low-energy electronic transitions. In the tetrasilanes 2-4, three distinct transitions are apparent. In tetrasilanes 5 and 6, MCD reveals the very weak transition that has been predicted to be buried under the first intense peak and to which the anomalous thermochromism of 6 and other short-chain oligosilanes has been attributed.     

Characterization of different high molecular weight angiotensinogen forms

BACKGROUND: Angiotensinogen is the substrate for renin in the system that releases angiotensin II. This renin-angiotensin system is an important regulator of blood pressure (BP), and defects in the system are linked to the development of hypertension. Native angiotensinogen is a 62,000-dalton monomer, but various high molecular weight forms also exist, which have not been well characterized. High molecular weight angiotensinogen has been reported to be 5% of the total angiotensinogen, and increases to 60% of the total during pregnancy and hypertension. The purpose of this investigation was to study high molecular weight angiotensinogen in normal plasma. METHODS: Normal human plasma was run on a gel filtration column, and high molecular weight angiotensinogen detected by Western blotting. Further purification was by ion-exchange chromatography. In vitro polymerization of angiotensinogen was analyzed by sodium dodecyl sulfate (SDS) and native gels. RESULTS: Two forms of high molecular weight angiotensinogen were found with molecular weights of 500,000 and 250,000 daltons on gel filtration, and 140,000 and 110,000 daltons, respectively, on nonreduced SDS-polyacrylamide gel electrophoresis, and at 62,000 daltons on reduced gels. Our estimation of the amount of high molecular weight angiotensinogen present is close to that reported previously. We also describe some of the in vitro polymerization characteristics of angiotensinogen, which can be explained by angiotensinogen being a member of the serpin family of proteins. CONCLUSIONS: The angiotensinogen polymers produced in vitro might provide a model system for some of the high molecular weight forms produced in vivo, and help in understanding their function.     

Increase in high molecular weight adiponectin by bariatric surgery-induced weight loss

Aim: To determine the changes in adiponectin multimers upon marked weight loss. Methods: Plasma samples were obtained preoperatively and 3, 6, 12 and 24 months after surgery from 12 obese subjects undergoing weight loss–inducing bariatric surgery. Seven non‐operated obese subjects served as controls. Plasma levels of adiponectin multimers were determined by protease digestion and Enzyme‐Linked ImmunoSorbent Assay (ELISA) detection. In addition, adiponectin multimers were assessed by western blotting. Results: In patients with weight loss after surgery but not in controls, total adiponectin and high molecular weight (HMW) adiponectin steadily increased during the observation period. Twenty‐four months after surgery, the increase in total and HMW adiponectin was 2.2 ± 0.46 and 1.4 ± 0.3 μg/ml, respectively. In contrast, plasma concentrations of middle and low molecular weight adiponectin remained unchanged. Conclusions: The increase in plasma adiponectin levels observed 24 months after bariatric surgery depended on continuous weight loss and was completely attributable to the HMW complex.     

Z-DNA: vacuum ultraviolet circular dichroism.

In concentrated salt or ethanolic solutions, the self-complementary copolymer poly(dG-dC).poly(dG-dC) forms a left-handed double-helical structure that has been termed "Z-DNA." The first evidence for this structure came from changes observed in the circular dichroism (CD) spectrum between 230 and 300 nm for low- and high-salt solutions (Pohl, F. M. & Jovin, T. M. (1972) J. Mol. Biol. 67, 675-696). In 3 M NaCl, the CD spectrum is approximately inverted compared to the B-form spectrum observed in low-salt solution. We measured the vacuum ultraviolet CD spectrum of poly(dG-dC).poly(dG-dC) down to 180 nm under conditions in which the 230- to 300-nm spectrum is inverted. Below 200 nm, where the B form exhibits the large positive peak at 187 nm that is characteristic of right-handed double-helical DNAs, the Z form exhibits a large negative peak at 194 nm and a positive band below 186 nm. Therefore, the Z-form vacuum ultraviolet CD spectrum resembles an inverted and red-shifted B-form spectrum. The magnitudes of the differences observed between the B and Z forms in the CD spectrum below 200 nm are about 10 times greater than those observed between 230 and 300 nm. The vacuum ultraviolet CD spectrum of poly(dG-dC).poly(dG-dC) in 3 M Cs2SO4 also is inverted compared to the B-form spectrum; however, between 230 and 300 nm, it is nonconservative with a negative maximum at 290 nm and a weak positive CD signal above 300 nm, presumably reflecting differential light scattering and indicating the existence of molecular aggregates. Our results suggest that the vacuum ultraviolet CD spectrum is sensitive to the handedness of double-helical DNA structures. The CD spectrum in this region should complement other spectroscopic methods in relating the structures of poly(dG-dC).poly(dG-dC) existing in solution to those determined in the solid state by x-ray crystallography.     

Changes of high molecular weight kininogen in various states

Changes of high molecular weight kininogen (HMW-K) clotting activity, antigen and cleavage in the plasma in the health and various diseases were studied. In 20 healthy individuals clotting activity of HMW-K, as measured by APTT one stage method, was 99 +/- 12% (male) and 84 +/- 15% (female). Antigen as measured by Laurell method were 106 +/- 24% (male) and 91 +/- 21% (female). In 35 patients with disseminated intravascular coagulation (DIC), both activity (78 +/- 33%) and antigen (69 +/- 31%) were statistically lower than those in normal individuals (p less than 0.01). In DIC both activity and antigen of HMW-K was correlated with serum albumin level. These results suggest that the cause of the lower level of HMW-K in DIC especially with septicemia is the result of lower production rather than consumption. In vivo cleavage of HMW-K was detected in plasma of a patient with septicemia and DIC by immunoblotting. The change of HMW-K was also assessed in other pathological states including liver cirrhosis, collagen disease, cardiopulmonary bypass and pregnant women.     

Human copper-containing superoxide dismutase of high molecular weight.

A superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1), distinct from previously known superoxide dismutases, has been isolated from human lung tissue. It is probably of the same nature as a previously demonstrated high molecular weight superoxide dismutating factor in human extracellular fluids. The enzyme has a molecular weight around 135,000 and is composed of four equal noncovalently bound subunits. Each molecule appears to have four copper atoms. No iron or manganese was found in the enzyme. Cyanide inhibits the enzyme efficiently. The enzyme brings about a first-order dismutation of the superoxide radical, the rate constant for the catalyzed reaction being about 1 X 10(9) M-1 s-1 per copper atom. The enzyme has hydrophobic properties. Affinity for various lectins indicates the presence of carbohydrate. Upon chromatography on heparin-Sepharose it is divided into three fractions, one with no, one with weak, and one with strong affinity for heparin.     

Conformation of cyclolinopeptide a observed by circular dichroism

A stereochemical investigation, by circular dichroism, of a synthetic nonapeptide (cyclolinopeptide A) in several organic and organic-sulfuric acid solvents is presented. From this examination, and results found for a conformationally rigid model compound, 1,7,7-trimethyl-3-azabicyclo [2.2.1] heptan-2-one(camphorolactam), it is concluded that cyclolinopeptide A may exist in several conformations in solution. None of these conformations is believed to be stabilized by intramolecular hydrogen bonds. Some details on an x-ray analysis of the cyclic nonapeptide are also presented.     

Phosphorylation of a high molecular weight DNA polymerase alpha.

Anti-human DNA polymerase alpha murine IgG SJK-287-38 [Tanaka, S., Hu, S.-Z., Wang, T. S.-F. & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390] neutralized DNA polymerase alpha activity from rat embryonic fibroblasts infected with a temperature-sensitive transformation mutant of Rous sarcoma virus (tsLA24). After centrifugation of a crude cytosol fraction from log-phase cells in a 5-20% linear sucrose gradient, polypeptides of Mr approximately equal to 185,000 and 220,000 were immunoprecipitated only from gradient fractions containing DNA polymerase alpha activity. When similar cultures were incubated in medium containing [32P]orthophosphate, it was found that the Mr 220,000 protein was phosphorylated but that the other peptides specific for polymerase alpha activity did not contain detectable amounts of phosphate. Phospho amino acid analysis of the high molecular weight immunoprecipitable proteins indicated that the labeled amino acid was phosphoserine. Incubation of 2.5 units of crude DNA polymerase alpha with 4 units of agarose-immobilized alkaline phosphatase resulted in a nearly complete inhibition of DNA polymerase alpha activity. Subsequent incubation of this preparation with 5 or 50 microM ATP, but not the nonhydrolyzable analog adenosine 5'-[gamma-thio]triphosphate, restored the in vitro DNA polymerizing activity. These results demonstrate that a high molecular weight DNA polymerase alpha (Mr approximately equal to 220,000) is phosphorylated in cultured cells and that this protein is a substrate for a serine kinase rather than the tyrosine-specific protein kinase of Rous sarcoma virus. The results suggest that phosphorylation/dephosphorylation reactions modulate the activity of this polymerase.     

Characteristics of high molecular weight insulins in insulinoma patients.

These studies were undertaken to characterize the high molecular weight immunoreactive insulins in plasma and tumor extracts of two patients with insulinoma, and to determine whether they are biosynthetic precursors of proinsulin. Plasma was chromatographed on a Sephadex G-50 column and fractions assayed for IRI. Three peaks were observed, one in the void volume region (6%), a peak in the proinsulin region (25%), and the largest peak comigrating with insulin standard (69%). Insulinoma slices were incubated for 4 h and high molecular weight IRI was observed in chromatographed extracts of the tumor, as well as in extracts of the incubation medium, which comprised up to 3% of the total IRI. Gel filtration chromatography of a pre-operative fasting plasma from the second patient revealed a heterogeneous distribution of IRI, with approximately 53% in the high molecular weight region. At surgery an insulinoma was removed. Slices were incubated for 4 h with 14C-amino acids and extracts chromatogrphed. 14C-labeled protein was observed in the V0, and in the proinsulin and insulin regions. These 14C-labeled proteins were precipitated with anti-insulin serum, and it was determined that whereas 60-90% of 14C-protein in the proinsulin-insulin region was precipitated, virtually none of the 14C-protein in the V0 was immunoprecipitated with anti-insulin serum. The specific activities (CPM precipitated by anti-insulin serum/muU IRI) were 1, 11.4, and 3.1 for V0, proinsulin, and insulin regions, respectively. These results suggest that proinsulin is synthesized first, followed by insulin, and then the high molecular weight IRI. The high molecular weight IRI is not a biosynthetic precursor of proinsulin. Rechromatography of the high-molecular weight IRI in the presence of 8M urea dissociated 90% into smaller immunoreactive components which chromatographed in the proinsulin and insulin region. It was concluded that high molecular weight IRI's are present in plasma, tumor extracts, and incubation medium from insulinoma patients. These high molecular weight IRI's are not biosynthetic precursors, but either aggregates of proinsulin and insulin, or insulin bound to larger proteins. These studies do not rule out the existence in humans of a smaller rapidly-turning over precursor to proinsulin similar to the pre-proinsulin discovered in a rat insulinoma by Chan et al.     

Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis

Deposition of aggregated protein into neurofilament-rich cytoplasmic inclusion bodies is a common cytopathological feature of neurodegenerative disease. How-or indeed whether-protein aggregation and inclusion body formation cause neurotoxicity are presently unknown. Here, we show that the capacity of superoxide dismutase (SOD) to aggregate into biochemically distinct, high molecular weight, insoluble protein complexes (IPCs) is a gain of function associated with mutations linked to autosomal dominant familial amyotrophic lateral sclerosis. SOD IPCs are detectable in spinal cord extracts from transgenic mice expressing mutant SOD several months before inclusion bodies and motor neuron pathology are apparent. Sequestration of mutant SOD into cytoplasmic inclusion bodies resembling aggresomes requires retrograde transport on microtubules. These data indicate that aggregation and inclusion body formation are mechanistically and temporally distinct processes.     

血清高分子量脂联素水平与动脉硬化的相关性

目的：探讨血清高分子量脂联素水平与动脉硬化的关系。方法：选择2011年1月至2011年12月到湘雅二医院进行健康体检的居家中老年人87例,收集其临床资料。以颈-股脉搏波传导速度（cf-PWV ）＝9 m/s为界分为：A组（cf-PWV＜9 m/s）21例, B组（cf-PWV≥9 m/s）66例,检测两组患者血压、血脂、血糖等代谢指标并进行比较。结果：与A组比较,B组血压明显升高,血低密度脂蛋白胆固醇、甘油三酯、总胆固醇水平显著升高,高密度脂蛋白胆固醇、血清总脂联素及高分子量脂联素水平显著降低（ P＜0.05或＜0.01）。多元回归分析显示血清高分子量脂联素（B＝-4.469, P＝0.011）、总脂联素（（B＝-3.96, P＝0.012）、高密度脂蛋白胆固醇（B＝-2.077, P＝0.015）水平和收缩压（B＝0.045, P＝0.045）是cf-PWV的独立预测因子。结论：血清高分子量脂联素和总脂联素水平可能是动脉硬化保护因素,其预测动脉硬化发生发展的作用值得进一步深入研究。     

Fenofibrate increases high molecular weight adiponectin in subjects with hypertriglyceridemia

Beneficial effects of peroxisome proliferator-activated receptor alpha (PPAR alpha) agonists have been reported in improving insulin sensitivity and raising serum total adiponectin. High molecular weight (HMW) adiponectin, which is secreted from adipocytes, and visfatin, which is also expressed in adipose tissue, is related to glucose metabolism. In view of the additive effects of PPAR alpha agonists on these adipocytokines and glucose metabolism, we investigated male hypertriglyceridemic subjects who were treated with fenofibrate. Eleven male subjects with hypertriglyceridemia were treated with fenofibrate and serum total cholesterol (T-cho), triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting glucose, fasting insulin, total and HMW adiponectin, and serum visfatin levels were determined before and 3 months after treatment. Fenofibrate treatment significantly lowered T-cho, triglyceride, and LDL-C levels. There was a statistically significant increase of HDL-C. No differences in insulin sensitivity indices (G/I ratio and HOMA-IR) were observed between before and after treatment with fenofibrate. The treatment did not alter the levels of serum total adiponectin and visfatin in the hypertriglyceridemic patients, while serum HMW adiponectin increased significantly. This study demonstrates that fenofibrate increases serum HMW adiponectin levels, whereas visfatin is not regulated by fenofibrate in hypertriglyceridemic subjects. Further investigations are warranted to determine whether the elevation of HMW adiponectin caused by fenofibrate might improve insulin sensitivity.