Presence Of Trp Research Articles

An anti-fouling wearable molecular imprinting sensor based on semi-interpenetrating network hydrogel for the detection of tryptophan in sweat

The challenge of heavy biofouling in complex sweat environments limits the potential of electrochemical sweat sensors for noninvasive physiological assessment. In this study, a novel semi-interpenetrating hydrogel of PSBMA/PEDOT:PSS was engineered by interlacing PEDOT:PSS conductive polymer with zwitterionic PSBMA network. This versatile hydrogel served as the foundation for developing an anti-fouling wearable molecular imprinting sensor capable of sensitive and robust detection of tryptophan (Trp) in complex sweat. The incorporation of PEDOT:PSS conductive polymer into the semi-interpenetrating hydrogel introduced diverse physical crosslinks, including hydrogen bonding, electrostatic interactions, and chain entanglement. This incorporation considerably boosted the hydrogel's mechanical robustness and imparted commendable self-healing property. At the same time, the synergistic coupling between the well-balanced charge of the zwitterionic network and the high conductivity of the PEDOT:PSS polymer facilitated efficient charge transfer. The formation of the desired molecular imprinting membrane of semi-interpenetrating hydrogel was triggered by self-polymerization of dopamine (DA) in the presence of Trp. The designed biosensor demonstrated good sensitivity, selectivity and stability in detecting the target Trp. Notably, it also exhibited exceptional anti-fouling abilities, allowing for accurate Trp detection in complex real sweat samples, yielding results comparable to commercial enzyme-linked immunoassay (ELISA).

Amino Acid‐Modulated Chirality Evolution and Highly Enantioselective Chiral Nanogap‐Enhanced Raman Scattering

AbstractControlling the growth and selection of chiral inducers is crucial for the generation of chiral inorganic structures as observed in nature. Herein, the plasmonic chirality evolution from the Au cube seed under the presence of L‐ or D‐amino acid as a chiral inducer is reported. The 432 Helicoid I structure is obtained using tryptophan (Trp), identical to the result with cysteine (Cys). The use of tyrosine (Tyr) produced a Helicoid IV‐type structure. However, no distinctive chiral structures can be obtained using phenylalanine (Phe), valine (Val) and leucine (Leu), which indicates the critical role of amino acids in chirality evolution. In particular, Trp‐Helicoid I nanoparticles (NPs) showed excellent enantioselective response toward L‐ or D‐Cys in the colorimetric assay and Raman analysis in the presence of Trp. Furthermore, the chiroptical property with a nanogap of 23.78 ± 0.82 nm in the Helicoid I structure further expands its applications for highly sensitive and quantitative chiral analysis for small molecules such as R/S‐epichlorohydrin (ECH), R/S‐limonene (LM), and R/S‐2‐butanol (BuOH) using a non‐polarized light source. The finding of amino acid‐dependent chirality evolution can widen the current understanding of chirality evolution in nature, and the use of helicoid structures with nanogaps incorporated with surface‐enhanced Raman scattering (SERS) can open a new avenue for chiral spectroscopic analysis.

Status of linusorbs in cold-pressed flaxseed oil during oxidation and their response toward antioxidants

Linusorbs (LOs, cyclolinopeptides) are a class of naturally occurring cyclic hydrophobic peptides found in flaxseed oil, whose oxidation states indicate the oxidative stability and bitterness of flaxseed oil. Subjected to 63 °C accelerated oxidation, most Met-containing LOs in cold-pressed flaxseed oil entirely depleted by the 6th day except CLP, and MetO2-containing LOs became the dominant ones. However, no MetO2 form of Trp-containing LOs, such as CLD, CLF and CLG, were detected. Given their oxidative kinetics, methionine sulfoxide (MetO) residue in some LOs was less sensitive toward oxidation in the presence of Trp (Tryptophan) group, and the oxidative stability of Met-containing LOs was CLP < CLB < CLL ≈ CLM < CLO, as compared to MetO-containing LOs: CLD < CLE < CLC < CLF ≈ CLG. When antioxidant was added into cold-pressed flaxseed oil to assess the additives’ antioxidant effect, no significant difference was observed on oil oxidative indices in early stage except α-tocopherol, where they vary dramatically in suppressing Met oxidation of LOs: L-AP (L-ascorbyl palmitate) > TBHQ (tert-butyl hydroquinone) > γ-tocopherol > carnosic acid > α-tocopherol. Besides its ability to suppress oxidation of Trp-containing LOs, L-AP also exhibits superior antioxidant effect on non-Trp-containing LOs due to its amphiphilic property. Due to the prooxidative effects of both α- and γ-tocopherol on LOs that contain Trp, it has been suggested that tocopherols may repair Trp residue on LOs, leading to increased tendency of MetO residues to oxidize. The findings of this research are critical for elucidating the antioxidative mechanism of LOs, which can further lead to the establishment of strategies in suppressing bitter after taste to produce high-quality flaxseed oil.

ТРАНСФОРМАЦІЯ ТРИПТОФАНУ В РЕАКЦІЇ З ТЕТРАХЛОРАУРАТНОЮ КИСЛОТОЮ

Gold nanoparticles (Au NPs) are effective agents for early diagnostic and treatment of a variety of diseases, including cancer. However, initial components in the synthesis like surfactants, which are commonly used to stabilize nanoparticles, can produce toxic effects on living organisms. Thus, finding ways to reduce the toxicity of nanoscale preparations is an actual problem. In this regard the essential aminoacid tryptophan (Trp) is a promising reagent for the synthesis of Au NPs that can serve as a reducer of Au(ІІІ) ions and particle stabilizer. In this work, in order to increase the biocompatibility of the nanoscale system, the synthesis of gold nanoparticles was performed in the presence of Trp. The reaction between HAuCl4 and Trp proceeded in aqueous medium with neutral pH at different temperatures. Spectral characteristics of products formed in Au/Trp systems and were studied. In the absorption spectra the main band of Trp at 280 nm was shifted to 250 nm during the reaction; for all studied systems upon excitation of fluorescence with the wavelength of λex = 340 nm the maximum of the emission bands were observed at λem = 450 nm, while for initial tryptophan (λex = 280 nm) it was located at λex = 375nm. Mass spectra of Trp solution in positive mode contained the signal at 131 Da, that was characteristic for ionized indole moiety of amino acid. For supernatants of studied Au/Trp systems in positive mode signals of ionized fragments with masses of 118, 146 and 174 Da were observed. Based on the data it was revealed that amino acid oxidation proceeded through the formation of kynurenine or through the "kynurenine pathway", that corresponds to the metabolic conversion of amino acid in human organism and proves the biocompatibility of formed products.

Mutation studies and structure-based identification of potential inhibitor molecules against periplasmic amino acid binding protein of Candidatus Liberibacter asiaticus (CLasTcyA)

Earlier reported crystal structure of CLasTcyA revealed unique features like relatively a larger substrate binding pocket, an extended C-terminal loop restricted by a disulfide bond and involvement of residues from hinge region in substrate binding. In present study, CLasTcyA mutants were created to evaluate the importance of these unique features through biophysical characterization. The Val58 in CLasTcyA was replaced by Trp, conserved in most cystine binding proteins, to reduce the size of the binding pocket. All other mutations were created in CLasTcyAV58W mutant as the presence of Trp could be used for intrinsic fluorescence studies. The CLasTcyAV58W showed a noticeable increase in binding affinity and thermal stability as compared to the native form. The mutation of two cysteines in triple mutant CLasTcyAV58W/C212S/C239S, removal of C-terminal extended loop in truncated CLasTcyAV58W/C212S and mutation of His95 from hinge region in the double mutant CLasTcyAV58W/H95A showed a marked decrease in stability-indicating the importance of the unique features in structure of CLasTcyA. The bioinformatics-based virtual screening was employed to screen the potential inhibitor molecules for detailed future studies. The results clearly establish the importance of unique features in structure-function relationship of CLasTcyA.

Ionpair-π interactions favor cell penetration of arginine/tryptophan-rich cell-penetrating peptides

Cell-penetrating peptides (CPPs) internalization occurs both by endocytosis and direct translocation through the cell membrane. These different entry routes suggest that molecular partners at the plasma membrane, phospholipids or glycosaminoglycans (GAGs), bind CPPs with different affinity or selectivity. The analysis of sequence-dependent interactions of CPPs with lipids and GAGs should lead to a better understanding of the molecular mechanisms underlying their internalization. CPPs are short sequences generally containing a high number of basic arginines and lysines and sometimes aromatic residues, in particular tryptophans. Tryptophans are crucial residues in membrane-active peptides, because they are important for membrane interaction. Membrane-active peptides often present facial amphiphilicity, which also promote the interaction with lipid bilayers. To study the role of Trp and facial amphiphilicity in cell interaction and penetration of CPPs, a nonapeptide series containing only Arg, Trp or D-Trp residues at different positions was designed. Our quantitative study indicates that to maintain/increase the uptake efficiency, Arg can be advantageously replaced by Trp in the nonapeptides. The presence of Trp in oligoarginines increases the uptake in cells expressing GAGs at their surface, while it compensates for the loss of charge interactions from Arg and maintains similar peptide uptake in GAG-deficient cells. In addition, we show that facial amphiphilicity is not required for efficient uptake of these nonapeptides. Thermodynamic analyses point towards a key role of Trp that highly contributes to the binding enthalpy of complexes formation. Density functional theory (DFT) analysis highlights that salt bridge-π interactions play a crucial role for the GAG-dependent entry mechanisms.

PHYSIOLOGICAL CHARACTERIZATION OF DIAZOTROPHIC BACTERIA ISOLATED FROM Brachiaria brizantha RHIZOSPHERE

ABSTRACT The objective of this study was to evaluate the ability of diazotrophic bacteria isolated from Brachiaria brizantha rhizosphere to grow at different pH values and salt concentrations, to produce indoleacetic acid (IAA), and to solubilize phosphate. Both acidity and salinity tolerance tests were performed on modified solid FAM medium. Acidity resistance tests were performed at pH values of 4.0, 5.0, 6.0, and 7.0, while, salt resistance was evaluated at concentrations of 0.01, 5.02, 9.99, 14.96, 19.98, 24.95, 29.98, 34.94, 39.97, 44.94, and 49.96 gL-1 NaCl. For the IAA production assay, bacterial strains were cultivated in liquid DYGS medium both in the absence and presence of tryptophan (Trp). In addition, phosphorus solubilization assay was performed in GL liquid medium. All strains grew at every pH value tested, and a high diversity was observed after salt resistance, IAA production, and phosphate solubilization testing. Strains UNIFENAS 100-51, UNIFENAS 100-52, UNIFENAS 100-60, UNIFENAS 100-63, and UNIFENAS 100-65 were those with the best growth at the highest salt concentrations. Furthermore, in the presence of Trp, strains UNIFENAS 100-63 and UNIFENAS 100-69 were the ones with the highest IAA production. Strain UNIFENAS 100-52 showed the best response to the in vitro phosphate solubilization assay. Based on these results, it can be seen that studies related to the physiological and metabolic characteristics of diazotrophic bacterial strains are important to ensure greater success in the field.

L-Tryptophan functionalized graphene quantum dots as a fluorescence indicator for pH detection in real water

L-Tryptophan (Trp) functionalized graphene quantum dots (Trp-GQDs) were prepared via a thermal cracking method, using Trp and citric acid (CA) as raw material. The as-obtained Trp-GQDs show stronger blue photoluminescence (PL) than simple graphene quantum dots (GQDs). The presence of Trp can obviously improve the luminescent properties of GQDs. The possible mechanism was investigated for the formation and luminescence properties of the Trp-GQDs. Furthermore, the Trp-GQDs exhibit a good liner relationship between fluorescence response and pH variation within the pH range of 4–12. More importantly, Trp-GQDs still perform well in sensitivity to pH values in real water samples. The results indicate that the probe shows great potential in pH detection.

Characterization of the binding of triprolidine hydrochloride to hen egg white lysozyme by multi-spectroscopic and molecular docking techniques

The interaction between triprolidine hydrochloride (TRP), a strong histamine H1-receptor antagonist and hen egg white lysozyme (HEWL) was investigatied by UV–vis, fluorescence, Förster's resonance energy transfer (FRET), circular dichroism (CD), Fourier transform infrared (FTIR) and molecular docking studies. The steady state fluorescence study shows that TRP quenches the intrinsic fluorescence of HEWL through a static quenching mode which also supported by UV–vis absorption study. The binding constant Kb was 2.31 × 104 M−1 and binding stoichiometry (n) was approximately one. The synchronous fluorescence spectroscopy showed that the binding of TRP changed the microenvironment around Trp and Tyr residue to a more hydrophilic region. The FRET analysis established that there is energy transfer from HEWL to TRP. The decreased α-helical content of HEWL on interaction with TRP indicated that TRP destabilized the native structure of HEWL. The alterations in the secondary structure of HEWL in presence of TRP is also revealed by 3D fluorescence and FTIR spectroscopic studies. In addition, molecular docking was performed to confirm the binding site of TRP on HEWL, mode of binding and amino acids involved in this binding. This study will provide an insight into the molecular basis of the interaction between HEWL and TRP at pharmacological level and highlights its significance in the clinical medicine.

Isolation of eupatorin (3′,5-dihydroxy-4′,6,7-trimethoxyflavone) from Albizia odoratissima and its application for l-tryptophan sensing

A simple fluorescence sensor for selective detection of L-Tryptophan (Trp) was developed by isolation of naturally occurring flavone Eupatorin (3′,5-dihydroxy-4′,6,7-trimethoxyflavone) (Eu) from stems of Albizia odoratissima plant for the first time. The Eu exhibited turn-on fluorescence response in presence of Trp in Dimethyl Sulphoxide (DMSO)/water medium. The 1:1 stoichiometry of Eu/Trp complex was confirmed by Job’s plot, Electrospray Ionisation (ESI) Mass Spectrometry (MS), and Density Functional Theory (DFT) calculation. The detection limit of the sensor reached 0.6 pM, superior to all previously reported data. We additionally used cyclic voltammetry and square wave techniques to confirm the mechanism of the developed sensor electrochemically. The potential application of the sensor was confirmed by detection of Trp in egg albumin and achieved very good recovery.

The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.

Biophysical insight into the binding of triprolidine hydrochloride to human serum albumin: Calorimetric, spectroscopy and molecular docking approaches

Triprolidine hydrochloride is extensively used as antihistamine and anticholinergic drug. We have examine the binding mechanism of triprolidine hydrochloride (TRP) with human serum albumin (HSA) using fluorescence spectroscopy, circular dichroism (CD), isothermal titration calorimetry (ITC) and molecular docking techniques. The fluorescence data demonstrated that TRP binds to HSA through static quenching. The thermodynamic parameters (∆H, ∆S and ∆G), binding stoichiometry and the binding constant between TRP and HSA were determined using ITC. Protein surface hydrophobicity of HSA has been calculated in presence and absence of TRP. CD results suggest the possibility of variation in the conformation of HSA in the presence of TRP. The binding site I was confirmed by molecular docking technique. The esterase-like activity of HSA shows that Arg-410 and Tyr-411 of sub-domain IIIA were directly involved in the binding process. Chemical unfolding study of HSA was carried out in the presence of TRP using GuHCl by CD and fluorescence spectroscopy.

Sự sản xuất IAA và siderophore của các dòng vi khuẩn liên hiệp thực vật và ảnh hưởng lên sự tăng trưởng của cây bắp (Zea mays L.) trồng trong chậu

Fifty five isolates of bacteria associated with maize were tested for the production of IAA using Fe-HClO4 and Fe-H2SO4 reagents. The production of IAA in the absence and the presence of 100 mg/L of trp was in the range of 0.24 to 9.99 and 0.14 to 12.51 mg/L, respectively. There were 16 isolates showing a remarkable decrease in the production of IAA in the presence of trp. Twelve isolates were further assayed for siderophore production on the CAS agar medium according to the method modified by Srivastava et al. (2013) but there was only one of them showing the ability. The length of the changed color edges starting from the borderline between the two media PS and CAS produced by isolate DDN10b changed from 1.30±0.10 cm at 2 DAI to 2.67±0.21 cm at 4 DAI. Effects of 5 potential isolates on plant growth of maize were studied under pots experiments. All isolates demonstrated an increase in shoot length, fresh shoot and root weight, and dry mass of one month old plants compared to the control. The best three were VTN2b (Bacillus subtilis), DDN10b (Burkholderia sp.) and TDB1 (Bacillus sp.) showing effects on dry mass equivalent to 50% NPK treatment or 75% NPK treatment and were suggested for field trials.

UVA illumination-induced optical coupling between tryptophan and natural dissolved organic matter.

Exposure of tryptophan (Trp) in aqueous solutions to UVA radiation resulted in decrease of Trp (C1) but generated an unknown fluorescent component (C2) with fluorescence emission maxima extending into wavelength range characteristic of humic substance (HS)-like material. The intensity of the two components (C1 and C2) could be operationally fit to linear functions of the illumination time t (0~40 h). However, C1 and C2 decreased and increased nonlinearly respectively in a mixture (Trp mixed with a reference sample of natural organic matter, i.e., NOM) which was exposed to the same UVA illumination, and the change of both C1 and C2 was faster than that in the absence of NOM. Moreover, the UV-Vis absorption maximum (ex = 278 nm) of Trp was faster removed for the mixture (after 5 h) than for Trp solutions without NOM (after 20 h). These observations suggested NOM-facilitated photobleaching of Trp and photoproduction of a new FDOM component under UVA illumination. Meanwhile, the fluorescence of the NOM in the absence of Trp was well represented by two HS-like components which decayed monotonically upon exposure to UVA light, while the photoinduced decay became nonmonotonic in the presence of Trp, and one component even increased with illumination during certain time window, indicating Trp-facilitated production of HS-like fluorescence signatures from NOM. The findings show that UVA-induced optical signature changes of tryptophan and HS-like materials are coupled and highlight the potential impact of absorption of solar UVA light by natural dissolved organic matter (DOM) on using the optical signatures to trace sources and sinks of DOM.

Poly (Rhodamine B) and MWCNTs Composite Film for the Separation and Simultaneous Voltammetric Quantification of Tryptophan, Paracetamol, Uric Acid, Dopamine and Ascorbic Acid

This paper focuses the separation and voltammetric quantification of five significant compounds, such as tryptophan (Trp), paracetamol (APAP), uric acid (UAC), dopamine (DA) and ascorbic acid (AA), which may coexist in urine samples. The poly (Rhodamine B) / MWCNTs composite film was created on glassy carbon electrode by cyclic voltammetric technique. Electroanalytical behaviors of analytes at poly (Rhodamine B)/ MWCNTs/GC electrode were examined using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Electrochemical impedance (EIS) and scanning electron microscopy (SEM) were employed to characterize the dielectric properties and morphology of the composites. The linear working ranges for simultaneous estimation of Trp, APAP, UAC, DA and AA were 2.0–100 μM, 3.0–70 μM, 2.0–100 μM, 0.5–20 μM, and 200–5000 μM, respectively and the detection limits were 0.25 μM, 0.65 μM, 0.54 μM, 0.05 μM, and 20 μM, respectively. The developed sensor was set up to be suitable for the routine analysis of the Trp, APAP, UAC, DA and AA in the urine and pharmaceutical samples and satisfactory results were received. Moreover, the sensor was applied for the quantification of p-aminophenol (PAP) in the presence of Trp, APAP, UAC, DA and AA. Well-separated oxidation peak currents were observed in phosphate buffer solution at pH 7.0.

A non-singlet oxygen mediated reaction photoinduced by phenalenone, a universal reference for singlet oxygen sensitization

Photosensitized oxidations involve the generation of radicals (type I mechanism) and/or the production of singlet molecular oxygen (1O2) (type II mechanism). Phenalenone (PN) is one of the well-known type II photosensitizers and is widely used as a 1O2 reference sensitizer. In this work, we have studied the interaction of tryptophan (Trp) with the excited states of PN, the production of 1O2 in the presence of Trp, the formation of radicals and the kinetics of the photodegradation of Trp under different experimental conditions. In contrast to what is usually assumed, we have proven that PN is also able to act as a type I photosensitizer. Moreover, we have demonstrated that the predominant mechanism of the photosensitization of Trp by PN involves an electron transfer initiated-process.

Minimalist probes for studying protein dynamics: thioamide quenching of selectively excitable fluorescent amino acids.

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond-a single atom substitution of the peptide backbone-can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetime measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a large number of measurements to model protein folding.

The Effect of Tryptophan on UV‐induced DNA Photodamage

Trp-DNA adducts resulting from UV irradiation of pyrimidine bases and nucleotides in the presence of tryptophan (Trp) have been the subject of previous research. However, the relative yield of the adducts compared with the UV screening effect of Trp has not been previously considered. To determine whether Trp-DNA adduct formation or absorption "screening" by Trp is the predominant process when DNA solutions are irradiated with UV light in the presence of Trp, we irradiated Trp-containing DNA oligonucleotide solutions with UVC light and incubated aliquots of those solutions with molecular beacons (MBs) to detect the damage. We observed a rapid decay of fluorescence of the MBs for pure DNA solutions, thereby indicating damage. However, in the presence of Trp, the fluorescence decay is prolonged, with time constants that increase exponentially with Trp concentration. The results are discussed in terms of a beneficial in vivo cellular protection rather than harmful adduct formation and suggest a net sacrificial absorption of UV light by Trp which actually protects the DNA from UV damage.

Restoration of Enzymatic Activity in a Ser-49 Phospholipase A2Homologue Decreases Its Ca2+-Independent Membrane-Damaging Activity and Increases Its Toxicity

Ammodytin L (AtnL) is a Ser-49 secretory phospholipase A2 (sPLA2) homologue with myotoxic activity. By analogy to the Lys-49 sPLA2 myotoxins, AtnL has been predicted to be enzymatically inactive due to the absence of the conserved Asp-49 that participates in coordination of the Ca2+ cofactor. By substituting Ser-49 and three other residues in the Ca2+-binding loop of AtnL, we obtained the first two enzymatically active mutants of Lys-49/Ser-49 sPLA2 homologues. The mutants LW and LV, which differed only by the presence of Trp and Val at position 31, respectively, efficiently hydrolyzed phospholipid vesicles, while recombinant AtnL displayed no activity. In contrast to AtnL but similarly to ammodytoxin A (AtxA), a homologous neurotoxic sPLA2, both mutants exhibited catalysis-dependent membrane-damaging ability, involving vesicle contents leakage and fusion. However, LW and LV also exhibited the potent, Ca2+-independent disruption of vesicle integrity characteristic of AtnL, but not of AtxA, in which leakage of the contents is not associated with membrane fusion. Although LV and, especially, LW have the advantage over AtnL of being able to act in both Ca2+-independent and Ca2+-dependent modes, and display higher cytotoxicity and higher lethal potency, they have a lower Ca2+-independent membrane-damaging potency and display reduced specificity in targeting muscle fibers in vitro. Our results indicate that, in evolution, Lys-49 and Ser-49 sPLA2 myotoxins have lost their Ca2+-binding ability and enzymatic activity through subtle changes in the Ca2+-binding network without affecting the rest of the catalytic machinery, thereby optimizing their Ca2+-independent membrane-damaging ability and myotoxic activity.

Post-translational Regulation of Human Indoleamine 2,3-Dioxygenase Activity by Nitric Oxide

The heme protein indoleamine 2,3-dioxygenase (IDO) is induced by the proinflammatory cytokine interferon-gamma (IFNgamma) and plays an important role in the immune response by catalyzing the oxidative degradation of L-tryptophan (Trp) that contributes to immune suppression and tolerance. Here we examined the mechanism by which nitric oxide (NO) inhibits human IDO activity. Exposure of IFNgamma-stimulated human monocyte-derived macrophages (MDM) to NO donors had no material impact on IDO mRNA or protein expression, yet exposure of MDM or transfected COS-7 cells expressing active human IDO to NO donors resulted in reversible inhibition of IDO activity. NO also inhibited the activity of purified recombinant human IDO (rhIDO) in a reversible manner and this correlated with NO binding to the heme of rhIDO. Optical absorption and resonance Raman spectroscopy identified NO-inactivated rhIDO as a ferrous iron (Fe(II))-NO-Trp adduct. Stopped-flow kinetic studies revealed that NO reacted most rapidly with Fe(II) rhIDO in the presence of Trp. These findings demonstrate that NO inhibits rhIDO activity reversibly by binding to the active site heme to trap the enzyme as an inactive nitrosyl-Fe(II) enzyme adduct with Trp bound and O2 displaced. Reversible inhibition by NO may represent an important mechanism in controlling the immune regulatory actions of IDO.