Phenolphthalein false-positive reactions from legume root nodules.

Presumptive tests for blood play a critical role in the examination of physical evidence and in the determination of subsequent analysis. The catalytic power of hemoglobin allows colorimetric reactions employing phenolphthalein (Kastle-Meyer test) to indicate "whether" blood is present. Consequently, DNA profiles extracted from phenolphthalein-positive stains are presumed to be from blood on the evidentiary item and can lead to the identification of "whose" blood is present. Crushed nodules from a variety of legumes yielded phenolphthalein false-positive reactions that were indistinguishable from true bloodstains both in color quality and in developmental time frame. Clothing and other materials stained by nodules also yielded phenolphthalein false-positive reactivity for several years after nodule exposure. Nodules from leguminous plants contain a protein (leghemoglobin) which is structurally and functionally similar to hemoglobin. Testing of purified leghemoglobin confirmed this protein as a source of phenolphthalein reactivity. A scenario is presented showing how the presence of leghemoglobin from nodule staining can mislead investigators.

Oxyleghemoglobin scavenges nitrogen monoxide and peroxynitrite: a possible role in functioning nodules?

It has been demonstrated that the NO* produced by nitric oxide synthase or by the reduction of nitrite by nitrate reductase plays an important role in plants' defense against microbial pathogens. The detection of nitrosyl Lb in nodules strongly suggests that NO* is also formed in functional nodules. Moreover, NO* may react with superoxide (which has been shown to be produced in nodules by various processes), leading to the formation of peroxynitrite. We have determined the second-order rate constants of the reactions of soybean oxyleghemoglobin with nitrogen monoxide and peroxynitrite. At pH 7.3 and 20 degrees C, the values are on the order of 10(8) and 10(4) M-1 s-1, respectively. In the presence of physiological amounts of CO2 (1.2 mM), the second-order rate constant of the reaction of oxyleghemoglobin peroxynitrite is even larger (10(5) M-1 s-1). The results presented here clearly show that oxyleghemoglobin is able to scavenge any NO* and peroxynitrite formed in functional nodules. This may help to stop NO* triggering a plant defense reaction.

Exploiting the conformational flexibility of leghemoglobin: a framework for examination of heme protein axial ligation.

We have exploited the intrinsic conformational flexibility of leghemoglobin to reengineer the heme active site architecture of the molecule by replacement of the mobile His61 residue with tyrosine (H61Y variant). The electronic absorption spectrum of the ferric derivative of H61Y is similar to that observed for the phenolate derivative of the recombinant wild-type protein (rLb), consistent with coordination of Tyr61 to (high-spin) iron. EXAFS data clearly indicate a 6-coordinate heme geometry and a Fe-O bond length of 185pm. MCD and EPR spectroscopies are consistent with this assignment and support ligation by an anionic (tyrosinate) group. The alteration in heme ligation leads to a 148mV decrease in the reduction potential for H61Y (-127+/-5mV) compared to rLb and destabilisation of the functional oxy-derivative. The results are discussed in terms of our wider understanding of other heme proteins with His-Tyr ligation.

[Isolation of multiple forms of metleghemoglobin reductase and leghemoglobin components from lupin nodules]. / Metod polucheniia mnozhestvennykh form metlegoglobinreduktazy i komponentov legoglobina iz kluben'kov liupina.

Method for simultaneous obtaining in homogeneous state of two main forms of metleghemoglobin reductase and main leghemoglobin components from lupine nodules was worked out. The method included steps of saturation with ammonium sulphate (40-80%), gel-filtration on Ultrogel AcA 44, isoelectric focusing and repeated isoelectric focusing. As result the forms of metleghemoglobin reductase with molecular weights 62 and 66 kDa were obtained purified 725 and 402 times respectively and obtained in homogeneous state. The total yield of activity was 37%. The form with 62 kDa molecular weight was more active.

Purification of leghemoglobin from nodules of Crotalaria infected with Rhizobium.

The leghemoglobin from nodules of Crotalaria juncea infected with Rhizobium spp. was purified to homogeneity. The protein was purified after precipitation with 40-80% (NH4)2SO4, and chromatography by anionic exchange and gel filtration. The leghemoglobin has a single component and showed an apparent M(r) of ca. 17,300 and 23,700 determined by SDS-PAGE and gel filtration, respectively. The amino acid composition showed that asparagine/aspartic acid, glutamine/glutamic acid, alanine, lysine, serine and leucine were the main amino acids. Iron was detected only in the band corresponding to the purified protein. The N-terminal amino acid sequence for the first 19 residues showed high similarities with several other leghemoglobins from other plants.

Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein.

Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.

Reaction of ferric leghemoglobin with H2O2: formation of heme-protein cross-links and dimeric species.

Ferric leghemoglobin in the presence of H2O2 is known to give rise to protein radicals, at least one of which is centred on a tyrosine residue. These radicals are quenched by at least two processes. The first one involves an intramolecular heme-protein cross-link probably involving the tyrosine radical; this leads to the formation of a green compound with spectral characteristics differing markedly from those of ferryl and ferric leghemoglobin. This green compound cannot be reduced by dithionite or ascorbate, precluding any role for this species as an oxygen carrier. It exhibits modified EPR and pyridine haemochromogen spectra, indicating that alterations occur at the porphyrin macrocycle level. The additional compound previously described [Puppo, A., Monny, C. and Davies, M.J. (1993) Biochem. J. 289, 435-438] appears to be a mixture of ferry Lb and this green compound. The second quenching route results in the formation of intermolecular cross-links and hence dimeric forms of the protein. Ascorbate and glutathione inhibit both this intermolecular dimer formation and the formation of the intramolecular haem-protein cross-links and are likely to play a protective role in vivo.

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha.

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.

Coordinated synthesis of leghemoglobin and root protein R18 in yellow lupin.

Uninfected roots of yellow lupin contain an abundant 18 kDa protein (referred to as R18), absent in the mature nodules. Some properties of this polypeptide are apparently similar to those of lupin leghemoglobins. However, the lack of any immuno crossreaction between R18 and leghemoglobin and differences in N-terminal amino acid sequences indicate that these proteins are coded by different genes. The decrease in the content of R18 protein in developing nodule is associated with the increased synthesis of leghemoglobin. This implies coordination of both events.

Sesbania rostrata root and stem nodule leghemoglobins: purification, and relationships among the seven major components.

By anion-exchange chromatography, the nitrogen fixing photosynthetic stem nodules and nonphotosynthetic root nodules of Sesbania rostrata are shown to contain the same seven major components of leghemoglobin (Lb), numbered LbI-LbVII in order of elution, although in different proportions. No novel component was found in photosynthetic nodules. All components of Sesbania Lb are monomeric, with molecular weights varying between 15,000 and 17,000, and at least six of them are separate gene products. It is suspected that variable conjugation with nonprotein moieties might be partially responsible for the molecular weight differences and anomalous behavior observed between isoelectric focusing and anion-exchange chromatography.

High-performance liquid chromatographic separation of leghemoglobins from soybean root nodules.

A crude fraction of soybean nodule ferri-leghemoglobin was absorbed onto a commercial DEAE HPLC column at pH 8.0, and resolved into eight isoprotein fractions. The identity of the leghemoglobins were determined by their order of elution from the DEAE column and by isoelectric focusing, using isoprotein standards isolated by conventional procedures. The three isoproteins of the c complex, c1, c2, c3, were not resolved. Unexpected heme containing proteins eluted just after leghemoglobin a and the c complex. These components possessed proteins similar to leghemoglobin a and the c complex, respectively, as judged by isoelectric focusing and absorbance spectra of the ferri and ferrous forms. The components designated leghemoglobin a' and leghemoglobin c' were also differentiated from leghemoglobin a and c by reverse-phase HPLC in a C18 column. Amounts of protein for the DEAE HPLC column ranged from 10 micrograms to 20 mg and sample volumes ranged from 2 to 250 microliters. The time required for chromatography varied depending on the gradient used, but never exceeded 40 min for samples up to 5 mg protein or 120 min for samples containing 5 to 20 mg protein. Due to the sensitivity of detection at 403 nm and leghemoglobins being the predominant chromophore at that wavelength, it was possible to quantitate levels of individual leghemoglobins in samples extracted from single nodules (ca. 15 to 65 mg fresh weight tissue). Quantitation was performed by interfacing the spectrophotometer output (10 mV) to a microcomputer and using commercially available software.

Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules.

Conformational changes induced by ligands and pH in lupine ferrileghemoglobin selectively modified at Tyr-E16 by the imidazolide spin label has been studied by the method of electron spin resonance in the pH range 6-13. It is shown that in the alkaline pH region the bound spin label registers a local conformational transition which precedes the alkaline denaturation of the protein. In aquamet, cyanide and nicotinate complexes of ferrileghemoglobin this transition occurs with pK 10.5, in acetate and azide complexes with pK 11. In all these ligand derivatives the transition is induced by alteration in the ionization state of one group (delta nH+ approximately equal to 1), most probably, the epsilon-amino group of Lys-GH3. The latter is linked with the Glu-A14 residue and this bond is essential for maintaining the native conformation of leghemoglobin. The ligand-induced conformational changes in the vicinity of the label are small and consist, most likely, in some alteration of the mutual arrangement of the AE and GH helical complexes. No correlation has been revealed between the spin state of the heme iron and the conformation of leghemoglobin in the region under study.

NMR studies of oxyleghemoglobin: assignment of distal histidine proton resonances and evidence for pH-dependent changes in conformation.

The resonance of the C-2 proton of the distal histidine has been assigned in the 400 MHz 1H-NMR spectrum of soybean ozyleghemoglobin a. This resonance is subject to a very large ring current shift from the heme and occurs to high field of the residual HO2H peak. The pH dependence was measured from a series of nuclear Overhauser effect difference spectra over a range of pH values. The resonance moves to high field with decreasing pH and reflects titration of a one proton-dissociable group with pK 5.5. Resonances of the heme substituents and distal amino acid side-chains are also sensitive to this titration. Changes in ring-current shifts and nuclear Overhauser effects indicate that a conformational change occurs in the heme pocket upon titration of the pK 5.5 group. We propose that protonation of the distal histidine with pK 5.5 is accompanied by movement of the imidazole ring towards the heme normal. This movement would allow interaction between the ligated oxygen molecule and the protonated distal histidine at acid pH.

[Properties of metlegoglobin reductase from lupine nodules]. / Metlegoglobinreduktaza kluben'kov liupina. Hekotorye svo¿istva.

The purification and properties of metlegoglobin reductase from lupine (Lupinus luteus L.) nodules are described. The purification procedure results in a 1056-fold purification of the enzyme with a total yield of 21%. The enzyme possesses the NADH-diaphorase activity. Metlegoglobin reductase is heterogenous during electrophoresis and isoelectric focusing. Electrophoresis produces two vicinal active bands, while isoelectrofocusing results in four active fractions. The fraction possessing the highest activity has a pI of 4.4. The enzyme is a flavoprotein, in which all flavins are represented by FAD. The molecular weight of the enzyme is 30 000. In some properties metlegoglobin reductase from lupine nodules is similar to methemoglobin reductase from erythrocytes and metmyoglobin reductase from muscles.

Separation and determination of the relative concentrations of the homogeneous components of soybean leghemoglobin by isoelectric focusing.

The multiple components of soybean ferric leghemoglobin are readily separated by analytical and preparative flat bed isoelectric focusing in both the presence and also the absence of the ligand nicotinate. In the presence of nicotinate the separation by isoelectric focusing is more rapid and results in sharper bands of the very stable ferric leghemoglobin nicotinate complexes. The separation is sensitive enough to permit analytical experiments on leghemoglobin from single nodules. Leghemoglobins a and c1 prepared by ion exchange chromatography are homogeneous by isoelectric focusing criteria. Leghemoglobin c2 prepared by ion exchange chromatography is an approximately 1:2 mixture of leghemoglobins c2 and c3. Leghemoglobin d consists of three components. The ratio of leghemoglobin a to leghemoglobin c3 content increases dramatically as very young nodules mature. The increase in relative leghemoglobin a content suggests that leghemoglobin a might be required for regulation of nodule O2 concentration only when the nodule structure is complex. The ratio of leghemoglobin c1 content to leghemoglobin c3 content increases somewhat during the early period of nodule development, while the ratio of leghemoglobin c2 content to leghemoglobin c3 content increases slowly throughout nodule development. Ratios of leghemoglobin b content to leghemoglobin a content and of total leghemoglobin d content to total leghemoglobin c content were almost independent of nodule age. Leghemoglobins a and b might be related biosynthetically, as might leghemoglobins c and d.

Leghaemoglobin from Trifolium subterraneum. Purification and characterization.

Leghaemoglobin from the subclover, Trifolium subterraneum cultivar Woogenellup, has been fractionated into at least four electrophoretically distinct components using the ion-exchange chromatographic procedure described by Appleby et al. (Appebly, C.A., Nicola, N.A., Hurrell, J.G.R. and Leach, S.J. (1975) Biochemistry 14, 4444--4450) for soybean leghaemoglobins. Unlike those of soybean, the subclover leghaemoglobins showed no evidence of autoxidation under identical isolation procedures, implying that these proteins have an unusually stable ferrous oxidation state. Circular dichroism in the far-ultraviolet (200--240 nm) indicated a high helicity (approx. 70%) as has been reported for other species of leghaemoglobins. However, circular dichroism in the near-ultraviolet region (240--300 nm) indicated that the haem-protein interactions may be considerably different in the subclover leghaemoglobins and this may explain their atypical resistance to autoxidation and the absence of nicotinate binding in these proteins.

[Heterogeneity of leghemoglobin from yellow lupine nodules]. / O geterogennosti leggemoglobinov iz kluben'kov zheltogo liupina.

A possibility is demonstrated to separate summary lupine leghemoglobins (which are salted out within 55--90% of ammonium sulphate saturation) into Lb I and Lb II components by means of ionic exchange chromatography on DEAE-cellulose. Lb I is eluted at lower ionic strength buffer than LbII, and differs from the latter in the form and the size of crystals. Both components have the same electrophoretic mobility and contain N-terminal glycine. LbII and LbI precipitate under gradual salting out within 55--75% and 78--90% of saturation respectively.