Cross-linking Of Subunits Research Articles

Gastric aggregation of micellar casein powders induced by high hydrostatic pressure: Effect of serum Ca2+ level

Micellar casein (MC) has a unique gastric colloidal behavior in response to Ca2+ cross-linking, and its aggregation properties are closely related to pepsin and gastric acid. In this study, MC with different levels of colloidal calcium phosphate (CCP) was obtained by high hydrostatic pressure (HHP) at different pressures, followed by spray drying to obtain the powders. Different amounts of calcium chloride (exogenous Ca2+) were added to MC powders prior to in vitro simulated digestion to investigate the effect of exogenous serum Ca2+ levels on the aggregation behavior and the structure change of curds generated in gastric tract. The results revealed that HHP induced the emergence of more Ca2+-binding sites, thus Ca2+ was more likely to bind to MC matrix with low CCP levels. Meanwhile, high serum Ca2+ level provided more opportunities to form aggregates. The Highest pressure (500 MPa) with the highest Ca2+ level (5 mM) caused the lowest solubility aggregates, which were only 30% at the end of gastric digestion (120 min), half of the control sample (0 MPa with 0.15 mM Ca2+). The results of wide-angle X-ray scattering / small-angle X-ray scattering suggested that both pepsin and gastric acid-induced aggregation via Ca2+ as a bridge. For pepsin, Ca2+ cross-linked between para-κ-casein; For gastric acid, Ca2+ recombined phosphorylation sites and caused cross-linking of casein subunits.

Effect of polysaccharides on the rheological behaviour of soy-wheat protein aggregation and conformational changes during high-moisture extrusion.

Due to the extrusion black box effect, polysaccharides determine the formation of meat-like fibrous structures by modulating the flow behaviour and structural changes of plant proteins under high-moisture extrusion conditions. However, the is limited knowledge on the mechanism of resolution. This study simulated the rheological properties of soy protein-wheat protein under 57% moisture conditions with addition of 4% sodium alginate (SA), 2% xanthan gum (XG), and 2% maltodextrin (MD). Effect of these polysaccharides on the aggregation behaviour and conformation of raw protein during high-moisture extrusion was investigated. It was revealed that the three polysaccharides were effective in increasing the interaction between proteins and between proteins and water. Among them, 4% SA elicited a significantly stronger storage modulus (gelation behavior) compared to the control. Analysis of different zones of extrudates by protein electrophoresis, particle size, and turbidity showed that SA-4% was able to form more high molecular protein aggregates (> 245 kDa) and promoted cross-linking of low molecular subunits (< 48 kDa), resulting in moderately sized protein aggregated particles. Fluorescence and UV spectra showed the transformation of protein tertiary structures in different extrusion zones, confirming that the key extrusion zone for protein conformational transformation by polysaccharides is the die-cooling zone. Furthermore, stretching of polypeptide chains and accelerated protein rearrangement facilitated the formation of more fibrillar structures. Theoretical support for polysaccharide modulation of plant protein quality in high moisture extruded products is provided by this study. This article is protected by copyright. All rights reserved.

N-Terminal Extensions Appear to Frustrate HU Heterodimer Formation by Strengthening Intersubunit Contacts and Blocking the Formation of a Heterotetrameric Intermediate

HU is a bacterial nucleoid-associated protein. Two homologues, known as HU-A, and HU-B, are found in Escherichia coli within which the early, late, and stationary phases of growth are dominated by HU-AA, HU-BB, and HU-AB dimers, respectively. Here, using genetic manipulation, mass spectrometry, spectroscopy, chromatography, and electrophoretic examination of glutaraldehyde-mediated cross-linking of subunits, in combination with experiments involving mixing, co-expression, unfolding, and refolding of HU chains, we show that the spontaneous formation of HU-AB heterodimers that is reported to occur upon mixing of wild-type HU-AA and HU-BB homodimers does not occur if chains possess N-terminal extensions. We show that N-terminal extensions interfere with the conversion of homodimers into heterodimers. We also show that heterodimers are readily formed at anticipated levels by chains possessing N-terminal extensions in vivo, when direct chain-chain interactions are facilitated through production of HU-A and HU-B chains from proximal genes located upon the same plasmid. From the data, two explanations emerge regarding the mechanism by which N-terminal extensions happen to adversely affect the conversion of homodimers into heterodimers. (1) The disappearance of the α-amino group at HU's N-terminus impacts the intersubunit stacking of β-sheets at HU's dimeric interface, reducing the ease with which subunits dissociate from each other. Simultaneously, (2) the presence of an N-terminal extension appears to sterically prevent the association of HU-AA and HU-BB homodimers into a critically required, heterotetrameric intermediate (within which homodimers could otherwise exchange subunits without releasing monomers into solution, by remaining physically associated with each other).

Inhibition and crosslinking of the selenoprotein thioredoxin reductase-1 by p-benzoquinone

Quinones are common in nature, and often cytotoxic. Their proposed toxicity mechanisms involve redox cycling with radical generation, and/or reactions with nucleophiles, such as protein cysteine (Cys) residues, forming adducts via Michael addition reactions. The selenenyl anion of selenocysteine (Sec) is a stronger nucleophile, more prevalent at physiological pH, and more reactive than the corresponding thiolate anion of Cys. We therefore hypothesized that Sec residues should be readily modified by quinones and with potential consequences for the structure and function of selenoproteins. Here, we report data on the interaction of p-benzoquinone (BQ) with the selenoprotein thioredoxin reductase-1 (TrxR1), which exposes an accessible Sec residue upon physiological reduction by NADPH. Our results reveal that BQ targets NADPH-reduced TrxR1 and inhibits its activity using 5,5′-dithiobis(2-nitrobenzoic acid) or juglone as model substrates, consistent with the targeting of both the Cys and Sec residues of TrxR1. In the absence of NADPH, BQ modified the non-catalytic Cys residues, leading to subunit crosslinking, mainly through disulfides, which also resulted in some loss of activity. This crosslinking was time-dependent and independent of the Sec residue. Addition of NADPH after BQ pre-treatment could resolve the disulfide-linked crosslinking. TrxR activity loss was also observed upon incubation of J774A.1 cells or cell lysates with BQ. These data suggest that BQ readily targets TrxR1, albeit in a rather complex manner, which results in structural changes and loss of enzyme activity. We suggest that TrxR1 targeting can explain some of the cytotoxicity of BQ, and potentially also that of other quinone compounds.

A structure-based mechanism of cisplatin resistance mediated by glutathione transferase P1-1

Cisplatin [cis-diamminedichloroplatinum(II) (cis-DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis-DDP-binding protein. Our results show that cis-DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.

Highly Contrasting Static Charging and Bias Stress Effects in Pentacene Transistors with Polystyrene Heterostructures Incorporating Oxidizable N,N'-Bis(4-methoxyphenyl)aniline Side Chains as Gate Dielectrics.

Charge storage and trapping properties of polymer dielectrics govern the charge densities of adjacent semiconductors and greatly influence the on-off switching voltage (threshold voltage, ) of organic field-effect transistors (OFETs) when the polymers are used as gate insulators. Intentional charging of polymer dielectrics in OFETs can change and affect the bias stress. We describe a chemical design and fabrication protocol to construct multilayer-stack dielectrics for pentacene-based OFETs using different polystyrene (PS)-based polymers in each layer, with oxidizable N,N-bis(4-methoxyphenyl)anilino (TPAOMe)-substituted styrene copolymers in arbitrary vertical positions in the stacks. Thermal, byproduct-free cross-linking of benzocyclobutene subunits provides integrity to the multilayer structure by preventing dissolution of the previous deposited layer. Neutron reflectivity data verified the multilayer morphology. We compared the shift before and after charging the stacks by application of ±100 V across 0.5-1 μm total film thicknesses. Bias stress was the dominant effect in bilayer devices with a TPAOMe layer in contact with the pentacene, indicated by the direction of shift associated with either polarity of external electric field. In structures with no TPAOMe subunit in contact with the pentacene, when charging with -100 V on top of the source and drain electrodes, electron injection from pentacene to dielectric was the major charging mechanism, again consistent with the bias stress direction. When charging with +100 V, bilayer devices without TPAOMe showed little change in , suggesting there was no bias stress effect or charge injection in these devices for this charging polarity. For the bilayer devices with the TPAOMe layer in the bottom, and the trilayer devices with TPOMe in the middle, when +100 V was applied, the shifts were opposite those expected from bias stress. Dipole formation or partial ionization of chargeable groups at the interface between the dielectric layers are likely polarization mechanisms in these cases. A simple analytical model levelports the plausibility of these mechanisms. This work provides examples of both stabilization and shifting of , and therefore controlling charge carrier density, in semiconductors overlying the dielectric multilayers.

Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis

L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity.

Probing the structure-function relationship of Mycobacterium leprae HSP18 under different UV radiations

Ultraviolet radiation, an effective sterilizing source, rapidly kills the causative organism (Mycobacterium leprae) of leprosy. But, the reasons behind this quick death are not clearly understood. Also, the impact of UV radiation on the antigen(s) which is/are responsible for the survival of this pathogen is still unknown. Many reports have revealed that M. leprae secrets a major immunodominant antigen, namely HSP18, whose chaperone function plays an important role in the growth and survival of this pathogen under various environmental insults. However, the effect of UV radiation on its structure and chaperone function is still unclear. Therefore, we have taken a thorough attempt to understand these two aspects of HSP18 under different UV radiations (UVA/UVB/UVC; doses: 1–50 J/cm2). Our study revealed that its chaperone function is decreased significantly with increasing doses of various UV radiations. These different UV irradiations perturb only its tertiary structure and induce tryptophan and tyrosine photo-oxidation to N-formyl kynurenine, kynurenine and dityrosine. Such photo-oxidation promotes the subunit cross-linking within a HSP18 oligomer, lowers the surface hydrophobicity and thermostability of the protein. All these factors together damage/reduce the chaperone function of HSP18 which may be an important factor behind the rapid death of M. leprae under UV exposure.

Wheat bran-associated subaleurone and endosperm proteins and their impact on bran-rich bread-making

The functionality of wheat bran-associated subaleurone and endosperm proteins was investigated. The wheat cultivars Apache and Akteur were used as they displayed widely different subaleurone protein contents upon microscopic analysis. Brushing of their roller mill bran yielded a peripheral endosperm- and subaleurone-enriched flour fraction with a protein content of 15.1% and 22.1% for Apache and Akteur, respectively, compared to 10.0% and 12.8% for regular flour. The peripheral endosperm fraction of Akteur, but not Apache, displayed increased glutenin and gliadin contents and increased glutenin subunit crosslinking. Bread-making using bran subjected to heat treatment and particle size reduction indicated that the gluten quantity and quality of the bran-associated tissue, as well as its availability and vitality, significantly affected dough and bread properties. These findings were validated using four commercial bran samples and demonstrate the potential importance of heat stabilization and wheat selection when producing bran for bread-making.

Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.

Synthesis, Fabrication, and Heterostructure of Charged, Substituted Polystyrene Multilayer Dielectrics and Their Effects in Pentacene Transistors

Charge trapping and storage in polymer dielectrics can be harnessed to control semiconductor devices. Organic transistor (OFET) gate insulators affect bias stress and threshold voltage (Vth), and charging them can preset the operating voltages and control bias stress. We describe a chemical design and film fabrication procedure for construction of stacks of polystyrene (PS) layers, each with arbitrary concentrations of potentially chargeable functional groups. Thermal cross-linking of benzocyclobutene subunits ensures layer integrity while keeping the layers free of polar functionality and small molecule byproducts. Neutron reflectivity (NR), scanning electron microscopy, and atomic force microscopy (AFM) showed that individual layer thicknesses varied systematically with polymer concentration in deposition solutions, and interfacial thicknesses ranged from 1.5 to 4 nm, independent of layer thickness, demonstrating formation of distinct layers with minimal roughness or intermixing. The PS-based materials ...

A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero-length oxidative crosslinking.

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) enzyme complex that upon activation by phosphorylation stimulates glycogenolysis. Due to its large size (1.3 MDa), elucidating the structural changes associated with the activation of PhK has been challenging, although phosphoactivation has been linked with an increased tendency of the enzyme's regulatory β-subunits to self-associate. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of β on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form β-β dimers in the nonactivated PhK complex. This peptide stimulated β-β dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of β competes with its counterpart region in the nonactivated enzyme complex in binding to the catalytic γ-subunit, we were able to formulate a structural model for the phosphoactivation of PhK. In this model, the nonactivated state of PhK is maintained by the interaction between the nonphosphorylated N-termini of β and the regulatory C-terminal domains of the γ-subunits; phosphorylation of β weakens this interaction, leading to activation of the γ-subunits.

Quantification of anti-aggregation activity of UV-irradiated α-crystallin

Ultraviolet radiation is a risk factor for cataractogenesis. It is believed that enhanced rates of lens opacification and cataract formation are the results of gradual loss of chaperone-like efficiency of α-crystallin upon exposure to UV light. To characterize chaperone-like activity of α-crystallin damaged by UV irradiation, a test system based on dithiothreitol-induced aggregation of holo-α-lactalbumin from bovine milk was used. The adsorption capacity of α-crystallin (AC0) with respect to the target protein (α-lactalbumin) was used as a measure of anti-aggregation activity of α-crystallin. The data on SDS-PAGE testify that UV irradiation of α-crystallin results in covalent cross-linking of subunits in α-crystallin oligomers. The dependence of AC0 value on the irradiation dose was compared with the UV-induced diminution of the portion of native α-crystallin estimated from the data on differential scanning calorimetry. On the basis of such comparison a conclusion has been made that the loss in chaperone-like activity is mainly due to UV-induced denaturation of α-crystallin subunits. Cross-linking of remaining native subunits leads to an additional decrease in anti-aggregation activity.

Chemical modifications of respiratory complex I for structural and functional studies.

Studies on chemical modifications of bacterial and mitochondrial complex I by synthetic chemical probes as well as endogenous chemicals have provided useful information on the structural and functional aspects of this enzyme. We herein reviewed recent studies that investigated chemical modifications of complex I by endogenous chemicals (e.g. Cys-S-nitrosation, Cys-S-glutathionylation, and Ser-O-phosphorylation) and synthetic reagents (e.g. Cys-SH modification by SH-reagents and the cross-linking of nearby subunits by bifunctional cross-linkers). We also reviewed recent photoaffinity labeling studies using complex I inhibitors, which can be recognized as "site-specific modification" by synthetic chemicals. In addition, we discussed the possibility of site-specific modification by various functional probes via ligand-directed tosylate (LDT) chemistry as a promising approach for unique biophysical studies on complex I.

Initial characterization of the FlgE hook high molecular weight complex of Borrelia burgdorferi.

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.

Emulsifying and Foaming Properties of Soy Protein Isolates with Covalent Modification by (-)-Epigallocatechin-3-Gallate

Soy Protein Isolates (SPI) with covalent modification by (-)-Epigallocatechin-3-Gallate (EGCG) were prepared under the alkaline condition. The effects of covalent modification on the emulsifying and foaming properties of SPI were evaluated. The Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) profiles of the modified SPI revealed that EGCG treatment caused cross-linking of subunits. Emulsifying activity of modified SPI significantly increased when compared with that of control (p<0.05) at the concentration of 35 mg/mL. Modification of SPI by EGCG caused a decrease in the foam volume initially. In the range of 15 to 35 mg/mL, the foaming activities of modified SPI were found to be less than those of the control (p<0.05). The foaming stabilities of modified SPI were significantly higher when compared to those of control (p<0.05). The results obtained in this study indicated the modification by EGCG resulted in the enhancement of emulsifying activity and foaming stability of SPI. This modification may serve as a promising approach for improving functional properties of SPI.

Spectroscopic and Kinetic Investigation of the Reactions of Peroxyacetic Acid with Burkholderia pseudomallei Catalase-Peroxidase, KatG

Catalase-peroxidases or KatGs can utilize organic peroxyacids and peroxides instead of hydrogen peroxide to generate the high-valent ferryl-oxo intermediates involved in the catalase and peroxidase reactions. In the absence of peroxidatic one-electron donors, the ferryl intermediates generated with a low excess (10-fold) of peroxyacetic acid (PAA) slowly decay to the ferric resting state after several minutes, a reaction that is demonstrated in this work by both stopped-flow UV-vis absorption measurements and EPR spectroscopic characterization of Burkholderia pseudomallei KatG (BpKatG). EPR spectroscopy showed that the [Fe(IV)═O Trp330(•+)], [Fe(IV)═O Trp139(•)], and [Fe(IV)═O Trp153(•)] intermediates of the peroxidase-like cycle of BpKatG ( Colin, J. Wiseman, B. Switala, J. Loewen, P. C. Ivancich, A. ( 2009 ) J. Am. Chem. Soc. 131 , 8557 - 8563 ), formed with a low excess of PAA at low temperature, are also generated with a high excess (1000-fold) of PAA at room temperature. However, under high excess conditions, there is a rapid conversion to a persistent [Fe(IV)═O] intermediate. Analysis of tryptic peptides of BpKatG by mass spectrometry before and after treatment with PAA showed that specific tryptophan (including W330, W139, and W153), methionine (including Met264 of the M-Y-W adduct), and cysteine residues are either modified with one, two, or three oxygen atoms or could not be identified in the spectrum because of other undetermined modifications. It was concluded that these oxidized residues were the source of electrons used to reduce the excess of PAA to acetic acid and return the enzyme to the ferric state. Treatment of BpKatG with PAA also caused a loss of catalase activity towards certain substrates, consistent with oxidative disruption of the M-Y-W adduct, and a loss of peroxidase activity, consistent with accumulation of the [Fe(IV)═O] intermediate and the oxidative modification of the W330, W139, and W153. PAA, but not H2O2 or tert-butyl hydroperoxide, also caused subunit cross-linking.

Haptoglobin Preferentially Binds β but Not α Subunits Cross-Linked Hemoglobin Tetramers with Minimal Effects on Ligand and Redox Reactions

Human hemoglobin (Hb) and haptoglobin (Hp) exhibit an extremely high affinity for each other, and the dissociation of Hb tetramers into dimers is generally believed to be a prerequisite for complex formation. We have investigated Hp interactions with native Hb, αα, and ββ cross-linked Hb (ααXLHb and ββXLHb, respectively), and rapid kinetics of Hb ligand binding as well as the redox reactivity in the presence of and absence of Hp. The quaternary conformation of ββ subunit cross-linking results in a higher binding affinity than that of αα subunit cross-linked Hb. However, ββ cross-linked Hb exhibits a four fold slower association rate constant than the reaction rate of unmodified Hb with Hp. The Hp contact regions in the Hb dimer interfaces appear to be more readily exposed in ββXLHb than ααXLHb. In addition, apart from the functional changes caused by chemical modifications, Hp binding does not induce appreciable effects on the ligand binding and redox reactions of ββXLHb. Our findings may therefore be relevant to the design of safer Hb-based oxygen therapeutics by utilizing this preferential binding of ββXLHb to Hp. This may ultimately provide a safe oxidative inactivation and clearance pathway for chemically modified Hbs in circulation.

Nitrative Stress Causes Nitration, Oxidation, and Subunit Cross Linking in Human Hemoglobin

AbstractHemoglobin (Hb) is the most abundant protein in human blood and we showed that under oxidative/nitrative stress conditions it is susceptible to cysteine oxidation, tyrosine nitration, and formation of a dimer of Hb subunits through tyrosine linkage. In the presence of hydrogen peroxide, Hb and its subunits efficiently convert nitrite into reactive nitrogen species, through reactions that are typical of peroxidases. If an exogenous phenolic substrate is present, Hb promotes its nitration with a fivefold higher efficiency with respect to the peroxidase‐like phenol coupling reaction. In the absence of an exogenous substrate, the protein itself undergoes covalent modification. Trypsin treatment of Hb modified under conditions mimicking pathophysiological conditions, followed byHPLC‐ESI‐MS/MS analysis, allowed detection of nitration of Yα24, Yα42, Yβ130 and Yβ145, and conversion of Cα104, Cβ93 and Cβ112 into cysteine sulfinic acids. As additional biomarkers of nitrative stress, we found a covalent dimer of Hb αβ subunits and covalently linked heme‐peptide. The dimer is selectively nitrated at Yα42. In a preliminary analysis of samples of human blood we found that low amounts of the dimer of subunits are always present. Therefore, if a correlation between the extent of Hb subunits coupling and pathological states could be established, this dimer could become an easily detectable biomarker of pathological conditions.

Stabilization of the hexameric glutamate dehydrogenase from Escherichia coli by cations and polyethyleneimine

The enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The stability of this enzyme was increased in the presence of Li+ in concentrations ranging from 1 to 10mM, 1M of sodium phosphate, or 1M ammonium sulfate. A very significant dependence of the enzyme stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of enzyme concentration on its stability was not significantly decreased by the presence of 10mM Li+. Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the enzyme subunits. This incubation does not have a significant effect on enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2h under conditions where the untreated enzyme retained only 20% of the initial activity, and the effect of the enzyme concentration on enzyme stability almost disappeared. This stabilization was maintained in the pH range 5–9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li+ on enzyme stability was maintained after enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the enzyme.