Oxygen binding to dithionite-reduced chloroperoxidase

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.     

红凤菜红色素水溶液的稳定性试验

从红凤菜（Gynura bicolor DC.）地上部分提取了红色素水溶液。在350－700nm范围内的扫描结果显示,该色素有2个吸收峰,位于543nm和598nm附近。在pH2.2-6.0的范围内色素较稳定。加热使其吸收峰值增大,但波长基本不变。0.2mmol/L Fe^3 引起色素溶液变色并产生大量沉淀;Al^3 也能使其产生沉淀;Cu^2 对该色素的影响较小;Zn^2 、对其无影响。氧化剂（H2O2）与还原剂（Na2SO3）均能使该色素褪色。光照对该色素无明显影响。     

The amino acid sequence of a ribosome-inactivating protein from Saponaria officinalis seeds

The complete primary structure of saporin SO-6, a ribosome-inactivating protein extracted from Saponaria officinalis seeds, has been determined. The sequence was reconstructed following purification and analysis of peptides obtained after digestion of the protein with different proteolytic agents. The protein is composed of 253 amino acids, corresponding to a molecular weight of 28,621 Da. Comparison of the primary structure of SO-6 with the sequence deduced from cDNA, shows amino acid substitutions in 11 positions, suggesting a tissue-related genetic variability. When the sequence of saporin is compared to those of two related proteins, ricin A chain and trichosanthin, a low degree of similarity (12%) is found; nevertheless some considerations about structure-function relationships and evolution of RIPs are possible.     

Spectroscopic and thermodynamic investigations on the binding of azure B to chondroitin sulfate and the structure of the metachromatic dye complex

The binding of azur B to chondroitin sulfate (CHS) was investigated using absorption spectroscopy. In aqueous solutions it is possible to distinguish three different dye species with absorption bands at 646, 597, and 555 nm. They are assigned to monomers, dimers, and higher aggregates of azure B, which become bound to CHS as the dye concentration (CD) increases. The short-wavelength band (555 nm) causes metachromasia in stained histological materials. When saturation occurs, the metachromatic azure B-CHS complex has a 1:1 composition, i.e., each anionic SO-4 and COO(-)-binding site of CHS binds one dye cation. The composition of the saturated metachromatic complex was determined by spectrophotometric and conductometric titration of CHS with azure B, while the SO-4 and COO- content of CHS was determined by conductometric titration of CHS-acid with NaOH. The binding isotherm of azure B to CHS was determined using gelpermeation chromatography. The isotherm can be described by the model of cooperative binding of ligands to linear biopolymers. We found good agreement between theoretical predictions and experimental findings in the range of 0 less than r less than 0.8 (r = the fraction of occupied binding sites). Using a Schwarz plot, we determined the binding constants of nucleation (Kn = 2.5 X 10(3) M-1) and aggregation (Kq = 1.2 X 10(5) M-1), as well as the cooperativity parameter (q = 50), T = 295 K. With increasing CD, the strong cooperativity of the dye binding favors the formation of metachromatic aggregates rather than monomers and dimers. From the temperature dependence of Kq we evaluated the standard binding enthalpy (delta Hoq = -20.0 kJ mol-1) and entropy (delta Soq = 29.7 JK-1 mol-1) of the cooperative dye binding. The binding was found to be strongly exothermic and accompanied by a thermodynamically favorable entropy increase, this being typical of hydrophobic interactions. Solid azure B-CHS complexes were prepared according to a special dialytic technique and were studied using a microspectrophotometer equipped with a polarizer and an analyzer. The metachromatic 1:1 complex has a broad, intense absorption band whose main peak occurs at 560 nm. This corresponds with the maximum of the metachromatic dye complex in aqueous solution, i.e. 555 nm. The CHS chains of the azure B-CHS complex can be mechanically aligned in a preferred direction (k). We were able to prepare excellently orientated and very fine dye-CHS films which were birefringent and dichroic - the more birefringent, the better the mechanical orientation.(ABSTRACT TRUNCATED AT 400 WORDS)     

红球菌SDUZAWQ对有机硫和无机硫的耐受性研究及脱硫基因的克隆

以筛选得到的红球菌SDUZAWQ为对象,研究其在不同浓度的有机硫化合物二苯并噻吩(DBT)存在下的脱硫能力,以及在0.2mmolLDBT和不同浓度Na2SO4同时存在下的脱硫情况。当DBT浓度高达6mmolL时,菌株仍能生长,而且检测出产物2-羟基联苯(2-HBP)的存在,说明该菌株具有耐受较高浓度DBT的能力。当DBT和Na2SO4同时存在时,DBT为菌株SDUZAWQ所利用,并且也检测出2-HBP,并非如文献所报道的红球菌在无机硫存在下不代谢DBT,表明该菌株能够耐受一定浓度的无机硫酸盐。对相关脱硫基因的克隆和测序结果显示,完整脱硫基因dszABC、其上游调控序列和dszD的序列与模式菌株RhodococcuserythropolisIGTS8的同源性分别是99%、100%和100%。     

Effects of acetazolamide on cerebrocortical NADH and blood volume

Acetazolamide (AZ), a potent carbonic anhydrase inhibitor in human and animal tissues, increases cerebral blood flow (CBF) by acidifying cerebral extracellular fluids. To demonstrate the relationship of increased CBF to brain O2 availability after AZ administration, a compensated fluorometer was used to study changes in the cerebrocortical redox balance in rabbits. Seven rabbits were anesthetized with pentobarbital sodium. Excitation light (366 nm) was conducted to the cerebrocortical surface of each animal by a 4-mm-diam fiberoptic light guide. Fluorescence emissions from cerebrocortical NADH (450 nm) were compared at different inspired O2 (FIO2) tensions. Reflected light (366 nm), which was used to determine a correction to the fluorescence signal, was separately quantitated and interpreted as an index of cerebrocortical blood volume. Reductions in FIO2 from 1.0 to 0.21, 0.14, 0.10, and 0.07 resulted in increases in both tissue blood volume and [NADH]. Intravenous AZ (25 mg/kg) increased cerebrocortical blood volume and reduced the [NADH], even during ventilation with 100% O2. The changes in brain redox balance caused by vasodilation with AZ were compared with those caused by vasodilatation with CO2. The NAD+/NADH redox state was a continuous function of FIO2 at all levels of arterial PCO2 (PaCO2), both before and after AZ administration. The improvement in cerebral O2 delivery caused by AZ-induced vasodilation was comparable to that caused by the vasodilatation that results from a PaCO2 elevation approximately equal to 12-15 Torr above normal. The slope of the relationship between [NADH] and FIO2 was similar at normal, low, and high levels of PaCO2. We conclude that AZ administration and PaCO2 elevation improve cerebral oxygenation by similar mechanisms.     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

棉铃虫感染中华卵索线虫后血淋巴酚氧化酶活性的变化及其分离纯化

研究了中华卵索线虫Ovomermis sinensis感染棉铃虫Helicoverpa armigera幼虫后宿主体内酚氧化酶活性的变化。研究结果表明,在感染后的第1天,中华卵索线虫的侵入引起酚氧化酶活性的增加,感染组酶活性是同期对照组的1.12倍; 但在随后的寄生期间,中华卵索线虫抑制了宿主的酚氧化酶活性,其中以第5天的抑制最为强烈： 同期对照组酶活性是感染组的1.52倍。对酚氧化酶进行了初步的分离纯化,纯化倍数为41.5倍,酶得率为12.7%,比活力为4 030.6 U/mg。     

Release of Citric Acid into the Medium by Aluminum-Tolerant Carrot Cells

One physiological characteristic of an Al-tolerant cell line(TA-1) selected from a cultured carrot cell line (SO-1) wasthe release of more citric acid into the medium than the parentalSO-1 line. Aluminum chloride was added to the media at a concentration,at which SO-1 as well as TA-1 could grow normally without inhibition.The amounts of citric acid and the soluble Al present in themedium were determined during the growth period. Much citricacid was released from TA-1 cells into the medium in the firsthalf of the culture period. At the time of maximum growth, theamount of citric acid in the medium of TA-1 cells was twiceas much as in the medium of SO-1 cells. The precipitates ofAl compound(s), which were formed in the medium by the additionof AlCl₃ as the Al source, became soluble as culture proceeded,depending on the amount of citric acid present in the medium. (Received September 3, 1983; Accepted May 9, 1984)     

Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies

A ribosome inactivating protein from Saponaria officinalis, SO-6, was purified and the N-terminus sequenced. The sequence shows extensive homology with Pokeweed antiviral protein, Pokeweed antiviral protein II, Pokeweed antiviral seed protein and dodecandrin. SDS gel electrophoresis in the Laemmli system revealed two bands of similar intensities with a smear between them, probably an artifact due to the high pI of the protein. Use of a harsher denaturing gel system resulted in one band in electrophoresis. Immune antisera was raised in rabbits against this protein and it cross reacted with other proteins (SO-5, SO-8 and SO-9) from seeds of Saponaria officinalis, but not with gelonin, Momordica charantia inhibitor and dianthin 32.     

Fluid Shear Induces Conformation Change in Human Blood Protein von Willebrand Factor in Solution

Many of the physiological functions of von Willebrand Factor (VWF), including its binding interaction with blood platelets, are regulated by the magnitude of applied fluid/hydrodynamic stress. We applied two complementary strategies to study the effect of fluid forces on the solution structure of VWF. First, small-angle neutron scattering was used to measure protein conformation changes in response to laminar shear rates (G) up to 3000/s. Here, purified VWF was sheared in a quartz Couette cell and protein conformation was measured in real time over length scales from 2-140 nm. Second, changes in VWF structure up to 9600/s were quantified by measuring the binding of a fluorescent probe 1,1′-bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate (bis-ANS) to hydrophobic pockets exposed in the sheared protein. Small angle neutron scattering studies, coupled with quantitative modeling, showed that VWF undergoes structural changes at G < 3000/s. These changes were most prominent at length scales <10 nm (scattering vector (q) range >0.6/nm). A mathematical model attributes these changes to the rearrangement of domain level features within the globular section of the protein. Studies with bis-ANS demonstrated marked increase in bis-ANS binding at G > 2300/s. Together, the data suggest that local rearrangements at the domain level may precede changes at larger-length scales that accompany exposure of protein hydrophobic pockets. Changes in VWF conformation reported here likely regulate protein function in response to fluid shear.     

Synthesis of a potential tetrasaccharide ligand for E-selectin

A potential tetrasaccharide ligand for E-selectin, (Na(+-)O(3)SO-3)Galbeta-(1-->4)[Fucalpha-(1-->3)]Glcbeta-(1-->6)Gal, an analogue of the ovarian cystadenoma glycoprotein tetrasaccharide fragment, was synthesized in a highly practical way.     

Binding interactions and conformational changes induced by sulfonated aluminum phthalocyanines in human serum albumin.

Phthalocyanines (Pc), which are extensively studied as tumor localizing photosensitizers for photodynamic therapy, are transported by the blood circulatory system to target tissues. Binding interactions between human serum albumin and differently sulfonated aluminum phthalocyanines (AlPcSn; n = 1-4) were studied using optical and ESR spectroscopy. AlPcSn (n = 1-3) occupy one strong binding site and eight weaker sites. The high affinity binding site interactions differ with respect to the degree of sulfonation and isomeric composition of the Pc. Phthalocyanines without SO-3 groups on adjacent iso-indole rings exhibit a high affinity binding site constant of K approximately 3-4 x 10(7) M-1, while Pc with two or three adjacent SO-3 groups show binding for this high affinity site that is no longer independent, but cooperative (alpha = 2), with K approximately 2-6 x 10(6) M-1. Binding isotherms for AlPcS4 and its close analog, tempoyl spin-labeled SL-AlPcS3, do not approach saturation at high ligand concentrations. Competition analyses between AlPcSn and spin-labeled fatty acids (5- and 16-doxyl stearate isomers) reveal that all compounds participate in cooperative (allosteric) interactions with the high affinity binding site of 16-DS, while extruding 5-DS isomer from certain sites and increasing the binding affinity for the remaining. Protein conformational dynamics was studied by ESR spectroscopy using covalent (alkylation of Cys34 residue) and noncovalent spin labeling (employing SL-AlPcS3). Phthalocyanines perturb conformational dynamics parameters (tauc and S) depending on the degree of sulfonation and isomeric composition corresponding to the type of sites, i.e., independent or cooperative, occupied on the HSA molecule.     

Solution structure of human von Willebrand factor studied using small angle neutron scattering

von Willebrand factor (VWF) binding to platelets under high fluid shear is an important step regulating atherothrombosis. We applied light and small angle neutron scattering to study the solution structure of human VWF multimers and protomer. Results suggest that these proteins resemble prolate ellipsoids with radius of gyration (R(g)) of approximately 75 and approximately 30 nm for multimer and protomer, respectively. The ellipsoid dimensions/radii are 175 x 28 nm for multimers and 70 x 9.1 nm for protomers. Substructural repeat domains are evident within multimeric VWF that are indicative of elements of the protomer quarternary structure (16 nm) and individual functional domains (4.5 nm). Amino acids occupy only approximately 2% of the multimer and protomer volume, compared with 98% for serum albumin and 35% for fibrinogen. VWF treatment with guanidine.HCl, which increases VWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales <10 nm without altering protein R(g). Treatment of multimer but not protomer VWF with random homobifunctional linker BS(3) prior to reduction of intermonomer disulfide linkages and Western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable intermonomer non-covalent interactions within the multimer. Overall, multimeric VWF appears to be a loosely packed ellipsoidal protein with non-covalent interactions between different monomer units stabilizing its solution structure. Local, and not large scale, changes in multimer conformation are sufficient for ADAMTS-13-mediated proteolysis.     

Changes in soil moisture drive soil methane uptake along a fire regeneration chronosequence in a eucalypt forest landscape

Disturbance associated with severe wildfires (WF) and WF simulating harvest operations can potentially alter soil methane (CH₄) oxidation in well‐aerated forest soils due to the effect on soil properties linked to diffusivity, methanotrophic activity or changes in methanotrophic bacterial community structure. However, changes in soil CH₄ flux related to such disturbances are still rarely studied even though WF frequency is predicted to increase as a consequence of global climate change. We measured in‐situ soil–atmosphere CH₄ exchange along a wet sclerophyll eucalypt forest regeneration chronosequence in Tasmania, Australia, where the time since the last severe fire or harvesting disturbance ranged from 9 to >200 years. On all sampling occasions, mean CH₄ uptake increased from most recently disturbed sites (9 year) to sites at stand ‘maturity’ (44 and 76 years). In stands >76 years since disturbance, we observed a decrease in soil CH₄ uptake. A similar age dependency of potential CH₄ oxidation for three soil layers (0.0–0.05, 0.05–0.10, 0.10–0.15 m) could be observed on incubated soils under controlled laboratory conditions. The differences in soil CH₄ uptake between forest stands of different age were predominantly driven by differences in soil moisture status, which affected the diffusion of atmospheric CH₄ into the soil. The observed soil moisture pattern was likely driven by changes in interception or evapotranspiration with forest age, which have been well described for similar eucalypt forest systems in south‐eastern Australia. Our results imply that there is a large amount of variability in CH₄ uptake at a landscape scale that can be attributed to stand age and soil moisture differences. An increase in severe WF frequency in response to climate change could potentially increase overall forest soil CH₄ sinks.     

Stopped-flow kinetic analysis of the bacterial luciferase reaction.

The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses.     

Binding of iodomercurates to sulfhydryl-blocked beta-lactoglobulin-A, -B, and -C

Sulfhydryl-blocked beta-lactoglobulins (beta-LG-S-SCH2CH2OH)-A, -B, and -C bind only one iodomercurate species, HgI3-, at only one site, with a dissociation constant of 4.0 X 10(-5) M at 25 degrees, pH 5.0, 0.10 ionic strength. (Binding to native beta-LG-SH-A, -B, and -C is more complex, involving the sulfhydryl and two other sites and several iodomercurates.) The red shift of the HgI3- spectrum on binding would ordinarily suggest a hydrophobic site, but the HgI3- site is distinct from, and independent of, the alkane-binding site of native and blocked beta-LG; HgI3- may bind a group that shifts its trigonal planar structure toward the tetrahedron of HgI4(2-). Binding of HgI3- to blocked beta-LG interferes with the well-known association of beta-LG-A to octamers at pH 4.6 and low temperature. The relation of the HgI3- site to the crystallographic iodomercurate-binding sites of beta-LG-SH is examined. To facilitate these and future studies of iodomercurate binding, the 200-400 nm spectra of HgI2, HgI3-, and HgI4(2-) in aqueous solutions and the thermodynamic formation constants at 25 degrees for the equilibria HgI2 + I- = HgI3- (4.9 X 10(3) M-1) and HgI3- + I- = HgI4(2-) (0.118 X 10(3) M-1) were obtained.     

Application of rapid-scanning, stopped-flow spectroscopy to the characterization of intermediates formed in the reactions of L- and D-tryptophan and beta-mercaptoethanol with Escherichia coli tryptophan synthase

The reactions of the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase with D- and L-Trp and the presteady-state reaction of L-Ser and beta-mercaptoethanol under different premixing conditions have been investigated by rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy. The reaction of alpha 2 beta 2 with L-Ser and beta-mercaptoethanol occurs in 3 detectable relaxations with rates similar to the 3 relaxations seen in the partial reaction with L-Ser and in the reaction with L-Ser and indole. The presteady-state phase of the reaction of beta-mercaptoethanol with the alpha-aminoacrylate intermediate is characterized by 2 relaxations. The RSSF spectra for this reaction show that the spectral changes that take place in these 2 phases are essentially identical. The L-Trp reaction is biphasic, and the spectral changes occurring in each phase of the reaction also are identical. The 2 new spectral bands formed (lambda max congruent to 420 nm and congruent to 476 nm) are assigned as the L-Trp external aldimine (Schiff's base) and L-Trp quinonoid intermediates, respectively. The reaction of D-Trp also is biphasic. Analysis of first and second derivatives of the RSSF spectral changes give evidence for the formation of spectral bands with lambda max of approximately 423 nm, approximately 450 nm, and approximately 478 nm. The positions and shapes of these bands suggest a D-Trp external aldimine structure (423 nm) and a quinonoidal species (450 and 478 nm). However, product studies do not support this latter assignment. The behavior of the D- and L-Trp reactions and the reaction of beta-mercaptoethanol with the alpha-aminoacrylate strongly indicate the pre-existence of 2 slowly equilibrating forms of the internal aldimine and of the alpha-aminoacrylate.     

An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.