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1.
The interaction of fatty acids (FA) with oxyhemoglobin (HbO2) was investigated. It was found that under effect of FA HbO2 is converted into the oxidized low-spin form, i. e., hemichrome. An increase in the level of reactive oxygen species was registered by the ESP method, using the spin traps, 1.2-dioxybenzo-3.5-disulphonate (tiron) and 5.5-dimethylpyrroline-1-oxide (DMPO). To investigate the nature of radical particles, the OH radical traps. thiourea and D-mannitol, as well as superoxide dismutase (SOD) and catalase were employed. The mechanism of interaction of HbO2 and FA is discussed; its feasible physiological role in the functioning of erythrocytes is postulated.  相似文献   

2.

Background

In forensic science, age determination of bloodstains can be crucial in reconstructing crimes. Upon exiting the body, bloodstains transit from bright red to dark brown, which is attributed to oxidation of oxy-hemoglobin (HbO2) to met-hemoglobin (met-Hb) and hemichrome (HC). The fractions of HbO2, met-Hb and HC in a bloodstain can be used for age determination of bloodstains. In this study, we further analyze the conversion of HbO2 to met-Hb and HC, and determine the effect of temperature and humidity on the conversion rates.

Methodology

The fractions of HbO2, met-Hb and HC in a bloodstain, as determined by quantitative analysis of optical reflectance spectra (450–800 nm), were measured as function of age, temperature and humidity. Additionally, Optical Coherence Tomography around 1300 nm was used to confirm quantitative spectral analysis approach.

Conclusions

The oxidation rate of HbO2 in bloodstains is biphasic. At first, the oxidation of HbO2 is rapid, but slows down after a few hours. These oxidation rates are strongly temperature dependent. However, the oxidation of HbO2 seems to be independent of humidity, whereas the transition of met-Hb into HC strongly depends on humidity. Knowledge of these decay rates is indispensable for translating laboratory results into forensic practice, and to enable bloodstain age determination on the crime scene.  相似文献   

3.
A spectrophotometric method is proposed for determining phospholipase A2 activity, which is based on the conversion of hemoglobin into hemichrome under the fatty acid action. The spectral difference between hemoglobin and hemichrome was registered by the difference spectrum with a minimum at 405 nm and a maximum at 423 nm. The absorption value determined as the difference between the spectrum maximum and minimum was proportional to the amount of the fatty acid derived from hydrolysis of phospholipids. The method enables the enzyme activity to be determined directly in the spectrophotometric cell.  相似文献   

4.
It has been found that superoxide radicals formed at autooxidation of Fe(II) ions or of their complexes with EDTA in the presence of NAD.H cause reduction of met-Hb into deoxy or oxy-form. Under the effect of hydroxyl radicals generated in the ultrasonic field in the presence of NADH reduction of met-Hb into the ferro-form (carboxy-Hb) was observed in Co atmosphere and not in O2 atmosphere. It was induced by a high oxidation rate of oxy-Hb by hydroxyl radicals into met-Hb as compared to carboxy-Hb. Reduction of met-Hb was shown to be accompanied with the formation of NAD+. The role of NAD. radicals in the reduction of met-Hb which acts as an antioxidant in respect to organic free radicals was discussed.  相似文献   

5.
Studies of fatty acid (FA) esterification by adipocytes have led to conflicting views with respect to how the process is regulated by norepinephrine (NE). It remains unclear whether NE directly modulates the pathway or whether its effects are indirect and reflect its well-known action to activate lipolysis. Changes in lipolysis can complicate estimation of esterification rates by altering both medium FA and the hydrolysis of newly formed FA esters. In this report, we describe an experimental approach that determined the effect of NE on FA esterification, amidst the complications introduced by activation of lipolysis. Esterification rates were estimated from the simultaneous incorporations (0.1-60 min) of [14C]glucose and [3H]oleate into diglyceride (DG), phospholipid (PL), and triglyceride (TG). Saturation kinetics of incorporation rates, with respect to FA, and more specifically to unbound or albumin-free FA (ubFA), were determined in both basal and NE-treated cells. To obtain true estimates of ester synthesis, incorporation rates were adjusted for label loss from breakdown of labeled esters. Our findings were: 1) In basal versus NE-treated cells, [3H]oleate, on its pathway to esterification, was diluted, respectively, by 2 and 50% of measured cell FA, and the diluting FA appeared derived from lipolysis. 2) Syntheses of PL, DG, and TG, estimated from incorporation of [14C]glucose, saturated at low ubFA. The Km for TG synthesis (0.06 microM) was within the physiological range of ubFA which meant that changes in plasma FA will modulate TG synthesis. PL synthesis, on the other hand (Km less than 0.01 microM), would be largely saturated under physiological conditions. 3) NE treatment increased the molar ratio of FA to albumin in the medium an average 8-fold and ubFA about 87-fold. In addition, NE accelerated hydrolysis of labeled PL and DG. Adjusting incorporation rates for these changes indicated that NE does not directly regulate glyceride synthesis. The assays described should allow estimation of glycerolipid synthesis under various metabolic or disease states and will distinguish direct effects from those reflecting changes in FA concentration or in hydrolysis of labeled FA esters.  相似文献   

6.
The stability of oxyhemoglobin S during mechanical shaking was enhanced by the addition of human serum albumin. The stabilizing effect was maximum when the concentration of serum albumin approached that of oxyhemoglobin, suggesting a molecular level interaction between them. The effects of serum albumin on oxyhemoglobin A were essentially similar to those on oxyhemoglobin S. Deoxy- and methemoglobins were also stabilized by serum albumin. The addition of human serum albumin to a solution containing sickle cell oxyhemoglobin slowly formed a compound which had an absorbance peak at 620 nm. After purification by Sephadex G-200 column chromatography, this compound was identified as methemalbumin. Comparison of the rates of formation of methemalbumin from hemoglobin with various ligand states and human serum albumin showed that the rate of formation from hemichrome was much faster than from met-, oxy- and deoxyhemoglobin. About 60% of the heme was transferred from hemichrome to albumin when the mixture was kept standing at room temperature for 5 min, in contrast to only 5% from methemoglobin. This result suggests that hemichrome, rather than methemoglobin, is the intermediate in the formation of methemalbumin from oxyhemoglobin and human serum albumin. This hypothesis is supported by the finding that the rate of formation of methemalbumin was faster at alkaline pH values than at acid pH values. Serum albumin from various animal sources showed different stabilizing effects. The formation of methemalbumin from these animal albumins was far less than that from human albumin.  相似文献   

7.
Nonenzymatic reduction of methemoglobin (met-Hb) is effectively catalyzed by NADH and riboflavin (RF). At low concentrations of met-Hb the EPR spectra of the ternary complex are characterized by an increased, whereas those at high met-Hb concentrations--by a decreased intensity of the RF semiquinone signal, which suggests that the met Hb reduction by a Leu-form of RF proceeds at a higher rate than that by the RF semiquinone radical. Riboflavin also accelerates the met-Hb reduction by a NADH-Fe2+ or NADH-Fe(2+)-EDTA mixture. It was found that the rate of met-Hb reduction by the organic radical increases in the following order: NAD. < RF H. < RF H2.  相似文献   

8.
S M Waugh  J A Walder  P S Low 《Biochemistry》1987,26(6):1777-1783
Early intermediates in the denaturation of hemoglobin, termed hemichromes, have been found previously to associate with the cytoplasmic domain of erythrocyte membrane band 3 in a manner which rapidly propagates into an insoluble, macroscopic copolymer. Because this interaction is thought to force a redistribution of band 3 in situ, the properties of the copolymerization reaction were investigated in greater detail. The band 3-hemichrome coaggregate was found to be stabilized largely by ionic interactions since elevation of either ionic strength or pH led to dissolution of the complex. The pH dependence, however, shifted to a more alkaline pH with increasing hemichrome concentration, suggesting a strong linkage between band 3 or hemichrome protonation and copolymer formation. The stoichiometry of the copolymer was measured at five globin chains per band 3 chain whenever underivatized dimer-tetramer hemichrome mixtures were employed. However, cross-linking of the hemichromes at either the alpha or the beta chains to form the stabilized tetramer yielded a copolymer stoichiometry of approximately eight globin chains per band 3 chain, i.e., two hemichrome sites per band 3 subunit. While underivatized hemichromes exhibited both a fast and slow phase of copolymerization, the cross-link-stabilized tetrameric hemichromes displayed predominantly the fast phase kinetics. Naturally occurring disulfide cross-linked hemichromes also reacted more avidly with band 3 than their reduced counterparts; however, the copolymerization process also proceeded to completion with totally reduced components. It is concluded that copolymerization of band 3 with hemichromes should occur under normal cellular conditions and at an accelerated velocity when the intracellular reducing power is low.  相似文献   

9.
Using chemiluminescence method, the oxidation level of lipids extracted from various organs and tissues of fish Coregonus peled (Gmelin) was investigated. The analysis of interrelation between the oxidation level, quantity and composition of phospholipids (PL), fatty acids (FA), natural antioxidants (NAO) was carried out. The oxidation level of the lipids under investigation is shown to increase in series: first go internal fat lipids, then--lipids of brain, white muscles, immature eggs, red muscles, liver. The lipid oxidation level was found to correlate with the quantity of PL, fraction content of phosphatidylethanolamine and cardiolipin, and concentration of the sum of polyunsaturated FA with the index of (20:5 divided by 22:6). It is shown that there is a regularity in the process of distribution in lipids both the sum of natural inhibitors, and individual AO, such as tocopherol, ubiquinone, ubichromenol, the quantity of which increases in accordance with the rising oxidation level of lipid substrate.  相似文献   

10.
分别对具4~5张完全叶桶栽甘蔗品种ROC22#和园林6#叶面喷施黄腐酸,分别为正常供水0(CK1)、0(CK2)、200、400、800、1200mg/L黄腐酸,研究在不同胁迫程度下不同浓度黄腐酸对甘蔗叶片叶绿素荧光参数、丙二醛(MDA)含量变化的影响。结果表明,经过黄腐酸处理后甘蔗叶片的PSⅡ原初光能转换效率(Fv/Fm)、光合量子产额(Yield)、光化学淬灭系数(qP)数值在中度和重度水分胁迫下都高于干旱对照CK2,品种和处理间参数的变化有差异;非光化学淬灭系数(qN)值则与正常供水对照比较没有大的差异。黄腐酸处理后甘蔗叶片MDA含量随处理浓度增大而降低,且明显低于干旱对照CK2。从2个参试品种6个处理浓度综合分析,水分胁迫下喷施黄腐酸可提高ROC22#和园林6#甘蔗苗期的抗旱性,以200、400mg/L处理效果较好。  相似文献   

11.
The equilibrium complexity involving different axially coordinated hemes is peculiar to hemoglobins. The pH dependence of the spontaneous exchange of ligands in the extracellular hemoglobin from Glossoscolex paulistus was studied using UV-Vis, EPR, and CD spectroscopies. This protein has a complex oligomeric assembly with molecular weight of 3.1 MDa that presents an important cooperative effect. A complex coexistence of different species was observed in almost all pH values, except pH 7.0, where just aquomet species is present. Four new species were formed and coexist with the aquomethemoglobin upon acidification: (i) a "pure" low-spin hemichrome (Type II), also called hemichrome B, with an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (ii) a strong g(max) hemichrome (Type I), also showing an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (iii) a hemichrome with unusual spin state (d(xz),d(yz))(4)(d(xy))(1) (Type III); (iv) and a high-spin pentacoordinate species. CD measurements suggest that the mechanism of species formation could be related with an initial process of acid denaturation. However, it is worth mentioning that based on EPR the aquomet species remains even at acidic pH, indicating that the transitions are not complete. The "pure" low-spin hemichrome presents a parallel orientation of the imidazole ring planes but the strong g(max) hemichrome is a HALS (highly anisotropic low-spin) species indicating a reciprocally perpendicular orientation of the imidazole ring planes. The hemichromes and pentacoordinate formation mechanisms are discussed in detail.  相似文献   

12.
两种抗旱剂对大叶黄杨光合特性的耦合效应   总被引:1,自引:0,他引:1  
通过田间试验,研究了FA旱地龙和BGA两种新型抗旱剂对大叶黄杨叶片光合特性的耦合影响.结果表明:FA旱地龙和BGA均能使大叶黄杨叶片的气孔导度(gs) 和蒸腾速率(Tr)缩小,净光合速率(Pn)和Pn/Tr增加;受BGA影响,FA旱地龙对未施用BGA叶片gs和Tr的抑制作用比对施用BGA叶片的抑制作用显著;FA旱地龙对未施用BGA叶片的Pn和Pn/Tr有促进作用,对施用BGA叶片的Pn和Pn/Tr有抑制作用;FA旱地龙对未施用BGA叶片的作用时间明显比对施用BGA叶片的作用时间长.BGA对FA旱地龙作用效果的影响原因可能与BGA和植物本身的光合特性有关.未施用BGA大叶黄杨适宜的FA旱地龙喷施浓度为2 g·L-1,FA旱地龙不适宜于施用BGA的大叶黄杨.  相似文献   

13.
Cell suspension cultures of potato (Solanum tuberosum, cv. Tamasha) were treated with fusaric acid (FA), a nonspecific fungal toxin produced by Fusarium species to study the effects of FA on H2O2 generation, lipid peroxidation, and activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and ascorbate peroxidase (APX). The toxicity of various FA doses was evaluated from viability of cultured cells of S. tuberosum. The toxic concentration of FA (10−3 M) reduced cell viability by 32% after 48-h incubation and induced alkalinization of the medium; the nontoxic concentration of FA (10−6 M) had no effect on cell viability and pH of the culturing medium. The treatment of cells with FA caused rapid reversible accumulation of H2O2 in cells, promoted lipid peroxidation, and elevated the activity of antioxidant enzymes. The toxic FA concentration elevated the intracellular H2O2 content by 51–59% and stimulated lipid peroxidation rate by 35–40%. The nontoxic FA concentration raised the H2O2 content by 84–91% and enhanced lipid peroxidation rate by 18–24%. The addition of FA induced transient biphasic induction of the antioxidant enzymes; the action of toxic and nontoxic concentrations differed in terms of the response amplitudes and dynamics. The results confirm the well-known toxic impact of high doses of FA on the cultured cells, which is determined by membrane transport disorders. In addition, the results reveal that toxic and nontoxic concentrations of FA are able to induce pro- and antioxidant systems in the cultured cells of S. tuberosum.  相似文献   

14.
Possible exogenous sources of formaldehyde and nitric oxide have been considered; the environment pollution conditions under which these compounds and their precursors have mutual effect on the organism; endogenous sources of FA and NO which are intermediates of the metabolism and key enzymes of their transformation (semicarbazide-sensitive amine oxidase and NO-synthase) the role of the C1 metabolic cycle pathways and methyl cycles in the FA formation and accumulation have been considered as well, various paths of FA toxic action have been characterized.  相似文献   

15.
The interaction of lysophospholipids with human oxy- and methemoglobin was studied. Anionic (acidic) lysophospholipids (lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylethanol, and lysophosphatidic acid) are potent effectors inducing the conversion of both forms of hemoglobin into hemichrome. Zwitterionic lysophospholipids (lysophosphatidylcholine, lysophosphatidylethanolamine, and lysosphingomyelin) did not influence oxyhemoglobin conversion, whereas methemoglobin conversion into hemichrome required much higher concentrations of these lysophospholipids compared to anionic lysophospholipids. Neutralization of negative charge on phosphate group of acidic lysophospholipids by Ca2+ was accompanied by partial or complete loss of their effector properties. The process of hemoglobin conversion to hemichrome is characterized by two isobestic points in the absorption spectra, indicating lack of stable intermediates. The present results are discussed in terms of the biological sense of the asymmetric distribution of phospholipids in the erythrocyte membrane.  相似文献   

16.
  • 1.1. Spectral analysis of the Soret region (450-350 nm) has shown that saturated fatty acids, alkyl ureas and urea induce the conversion of methemoglobins A, S, and F to the hemichrome state.
  • 2.2. In the presence of fatty acids (C8-C16), methemoglobin F is converted to the hemichrome state more readily than either methemoglobins A or S.
  • 3.3. Using several alkyl ureas (methyl, ethyl, propyl, butyl), the extent of hemichrome formation was as follows: met Hb F > met Hb S > met Hb A. The ability of these compounds to induce hemichrome formation is related to their increasing hydrophobicity.
  • 4.4. Conversion to the hemichrome state in the presence of urea (5M) led to the formation of molecular aggregates of hemoglobins S, F and A which may be initated by subunit dissociation and conformational changes, coupled to increased globin-globin interactions.
  • 5.5. Similar aggregation occurred for methemoglobin S in the presence of octanoic acid; no significant aggregation was evident for methemoglobin A after 10 hr of exposure to octanoic acid.
  相似文献   

17.
The interaction of sodium dodecyl sulfate (SDS) at a concentration range (0-515 microM) below the critical micelle concentration (CMC approximately 0.83 mM) with human native and cross-linked oxyhemoglobin (oxyHb) and methemoglobin (metHb) has been investigated by optical spectroscopy and stopped-flow transient kinetic measurements. It is observed that the interaction of SDS with human native and cross-linked oxyHb shows the disappearance of the bands of oxyHb at 541 and 576 nm and the appearance at 537 nm. The resultant spectra are characteristic of low spin (Fe(3+)) hemichrome. Similarly SDS has been found to convert human native and cross-linked high spin (Fe(3+)) metHb to low spin (Fe(3+)) hemichrome. The interaction of SDS with oxyHb suggests a conformational change of the protein in the heme pocket, which may induce the binding of distal histidine to iron leading to the formation of superoxide radical. The formation of hemichrome from metHb is found to be concentration-dependent with SDS. The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. The interaction of SDS with human cross-linked oxy and met hemoglobin shows results similar to those for human native oxy and met hemoglobin indicating that the covalent modification does not alter the interaction of SDS with cross-linked hemoglobin.  相似文献   

18.
Mitochondrial aconitase has been shown to be inactivated under the effects of many compounds and critical states. Fluoroacetate (FA) is the best-known aconitase-inhibiting toxic agent. The biochemistry of the toxic action of FA has been rather well studied; however, no effective therapy has been developed over the past six decades. To search for new approaches to the development of possible antidotes, experiments were carried out in vitro with rat liver mitochondria, Ehrlich ascite tumor (EAT) cells, and cardiomyocytes exposed to FA or fluorocitrate (FC). FA produced its effects at much higher concentrations as compared with FC; in experiments with mitochondria these effects depended on respiratory substrates: with pyruvate, FA induced a slow oxidation and/or a leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides (PN) was prevented by the incubation of mitochondria with cyclosporin A. Studies of the PN level and dynamics of Ca2+ in EAT cells during activation by ATP also revealed the PN leak from mitochondria, which led to a shift in the balance of mitochondrial and cytosolic NAD(P)H under action of FA. Moreover, an increase of cytosolic Ca2+ was revealed in the cells exposed to FA, which could be explained by the activation of plasma membrane calcium channels. This mechanism could affect the amplitude and rate of calcium waves in cardiomyocytes under the effects of FA. We emphasize the reciprocal relationship between intracellular PN dynamics and calcium balance and discuss possible pathways of metabolic modulation in the context of development of effective therapy of poisoning with FA and other aconitase inhibitors.  相似文献   

19.
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.  相似文献   

20.
We analyzed the developmental time, egg-to-adult viability, and developmental stability (fluctuating wing size asymmetry) in Drosophila subobscura, maintained for six generations on different concentrations of lead. Development time is significantly affected by generation and lead concentration, but interaction of these factors is not a significant source of variability for this fitness component. Generation and the interaction generation x concentration of lead significantly affect egg-to-adult viability. Levene's test of heterogeneity of variance showed that variability of FA is not significant in any of the samples. Within both lead concentrations females showed significantly higher FA indices for the wing width than males. Within sexes, a significantly higher FA was found only in females for wing width FA between the control and the lower concentration of lead. The results show that if strong relationship between FA and the studied fitness components exists, it results in a stronger selection of unstable genotypes under lead as a stress factor and, consequently, FA needs to be used with caution as a biomarker in natural populations under environmental stress.  相似文献   

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