Presence Of L-Phe Research Articles

Preparation of Molecularly Imprinted Electrochemical L-Phenylalanine Sensor with p-Toluene Sulfonic Acid Modified Pt Electrode

L-Phenylalanine (L-Phe) sensors were prepared with two steps by modifying the Pt electrode using the molecular imprinting technique. In first step, polymeric films based on p-toluene sulfonic acid (PTSA) were electropolymerized on the Pt electrode in the presence of L-Phe as template molecule. In the second step, L-Phe, the template molecule, was desorbed from the film structure coated on the electrode surface using acid. The surface properties and morphologies of the prepared modified electrodes were checked by SEM analysis technique. These modified electrodes were used for determination of L-Phe by differential pulse voltammetry (DPV). Electrodes obtained by molecular imprinting method showed R2 = 0.9997 in the concentration range of 2–2000 μM L-Phe. The limit of detection (LOD) of L-Phe selective electrodes was found as 0.59 μM and the limit of quantification (LOQ) was 1.77 μM. The first three repetition result with the same modified electrode was observed as 96.83% and the RSD value was 14.96% for ten replicates. For twenty modified electrodes, reproducibility was obtained as 97.67% stable between first electrode and twentieth electrode, and the RSD value was observed to be 2.33%. According to these results, prepared molecular imprinted electrodes have high repeatability, stability and selectivity for the determination of L-Phe.

L-phenylalanine-imprinted polydopamine-coated CdS/CdSe n-n type II heterojunction as an ultrasensitive photoelectrochemical biosensor for the PKU monitoring

A simple and highly sensitive photoelectrochemical biosensor towards L-phenylalanine, as a kind of typical essential amino acid and phenylketonuria biomarker was developed on a surface molecular imprinted (MIP) polydopamine-coated CdS/CdSe/Zn heterojunction. Hierarchical marigold flower-like Zn layer decorated by n-type dichalcogenides interfacial heterojunction was successfully designed and synthesized on Ti foil for PEC converter by in situ electrodeposition. A visible-light-driven molecular imprinting film was prepared through the electropolymerization of dopamine in the presence of L-Phe as biomarker. The combination of bio-MIP and photoelectrochemistry overcomes the defects of the PEC method, which is the absence of selectivity, and offers a new PEC sensor with high sensitivity and selectivity based on visible-light-driven heterojunction and biopolymer-enhanced strategy. The unique interfacial between the Zn marigold flower layer as low work function support and CdS/CdSe n-n heterojunction as well as n-type characteristics of polydopamine imprinted by L-Phe biomarker drastically increase the light trapping and absorption in the visible range, and dramatically inhibit the charge carrier recombination, which is crucial for boosting the Bio-PEC activity. Photocatalytic, electrocatalytic and physicochemical properties of the above-mentioned layers were fully characterized. As-prepared PEC biosensor displayed superb performance for the detection of L-Phe biomarker in the optimized condition obtained from central composite design modeling, showing two linear range 0.005–2.5 and 2.5–130 μM and a low detection limit of 0.9 nM. This work suggests that such L-Phe-imprinted polydopamine-coated Zn/CdS/CdSe heterojunction is greatly promising for being applied in photoelectrochemical biosensing with high photo-electron conversion efficiency.

Antagonist Effects of l-Phenylalanine and the Enantiomeric Mixture Containing d-Phenylalanine on Phospholipid Vesicle Membrane.

One of the congenital flaws of metabolism, phenylketonuria (PKU), is known to be related to the self-assembly of toxic fibrillar aggregates of phenylalanine (Phe) in blood at elevated concentrations. Our experimental findings using l-phenylalanine (l-Phe) at millimolar concentration suggest the formation of fibrillar morphologies in the dry phase, which in the solution phase interact strongly with the model membrane composed of 1,2-diacyl-sn-glycero-phosphocholine (LAPC) lipid, thereby decreasing the rigidity (or increasing the fluidity) of the membrane. The hydrophobic interaction, in addition to the electrostatic attraction of Phe with the model membrane, is found to be responsible for such phenomena. On the contrary, various microscopic observations reveal that such fibrillar morphologies of l-Phe are severely ruptured in the presence of its enantiomer d-phenylalanine (d-Phe), thereby converting the fibrillar morphologies into crushed flakes. Various biophysical studies, including the solvation dynamics experiment, suggest that this l-Phe in the presence of d-Phe, when interacting with the same model membrane, now reverts the rigidity of the membrane, i.e., increases the rigidity of the membrane, which was lost due to interaction with l-Phe exclusively. Fluorescence anisotropy measurements also support this reverse rigid character of the membrane in the presence of an enantiomeric mixture of amino acids. A comprehensive understanding of the interaction of Phe with the model membrane is further pursued at the single-molecular fluorescence detection level using fluorescence correlation spectroscopy (FCS) experiments. Therefore, our experimental conclusion interprets a linear correlation between increased permeability and enhanced fluidity of the membrane in the presence of l-Phe and certifies d-Phe as a therapeutic modulator of l-Phe fibrillar morphologies. Further, the study proposes that the rigidity of the membrane lost due to interaction with l-Phe was reinstated-in fact, increased-in the presence of the enantiomeric mixture containing both d- and l-Phe.

L-Amino Acids Promote Calcitonin Release via a Calcium-Sensing Receptor: Gq/11-Mediated Pathway in Human C-Cells.

Human calcitonin release is promoted by elevated extracellular Ca2+ (Ca2+o) concentration acting, at least in part, via the calcium-sensing receptor (CaSR). The CaSR is positively modulated by L-amino acids, including the aromatic amino acids L-phenylalanine (Phe) and L-tryptophan (Trp). To investigate the effect of L-amino acids on human calcitonin secretion, we selected thyroid TT cells and exposed them to various Ca2+o concentrations in the absence or presence of L-Phe, plasma-like mixtures of L-amino acids, or the clinically effective positive modulator (calcimimetic) cinacalcet. In the presence of L-Phe or plasma-like mixtures of amino acids, TT cells exhibited enhanced Ca2+o sensitivity in assays of calcitonin release and intracellular Ca2+ mobilization. Furthermore, the effect of elevated Ca2+o and L-Phe on calcitonin release was markedly suppressed by the calcilytic NPS-2143. These effects were dependent on CaSR-mediated activation of Gq/11 as revealed by the specific inhibitor YM-254890. The findings support the hypothesis that calcitonin release is stimulated by increases in plasma L-amino acid levels as well as elevated Ca2+o concentration. They also demonstrate that stimulated calcitonin release as well as basal levels of calcitonin secretion are mediated by a CaSR:Gq/11 signaling mechanism.

Dual-Signal Electrochemical Enantiospecific Recognition System via Competitive Supramolecular Host-Guest Interactions: The Case of Phenylalanine.

For developing highly selective and sensitive electrochemical sensors for chiral recognition, taking advantage of the synthetical properties of β-cyclodextrin (β-CD, strong host-guest recognition) and carbon nanotubes wrapped with reduced graphene oxide (CNTs@rGO, excellent electrochemical property and large surface area), as well as the differences in binding affinity between β-CD and guest molecules, a dual signal electrochemical sensing strategy was proposed herein for the first time in chiral recognition based on the competitive host-guest interaction between probe and chiral isomers with β-CD/CNTs@rGO. As a model system, rhodamine B (RhB) and phenylalanine enantiomers (d- and l-Phe) were introduced as probe and target enantiomers, respectively. Due to the host-guest interactions, RhB can enter into the β-CD cavity, showing remarkable oxidation peak current of RhB. In the presence of l-Phe, competitive interaction with the β-CD cavity occurs and RhB are replaced by l-Phe owing to the stronger binding affinity between l-Phe and β-CD, which results in the peak current of RhB decreasing and the peak current of l-Phe appears, and interestingly, the changes of both signals linearly correlate with the concentration of l-Phe. As for d-Phe, it cannot replace RhB owing to the weaker binding affinity between d-Phe and β-CD. Based on this, a dual-signal electrochemical sensor was developed successfully for recognizing Phe. This dual-signal sensing strategy can provide highly selective and sensitive recognition compared to single-signal sensor and has important potential applications in chiral recognition.

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease-Associated “Toggle” Calcium-Sensing Receptor Mutations

The Ca2+-sensing receptor (CaSR) regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+ ([Ca2+]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.

Photosensitization by phenylalanine of carboxylic acids, amides, and peptides in frozen aqueous solution.

Abstract— In order to elucidate the mechanism of the photosensitization of proteins and peptides by aromatic amino acids, the behaviour of aliphatic carboxylic acids and amides upon irradiation in frozen aqueous solution in the presence of phenylalanine (l-Phe) has been studied by EPR spectroscopy. EPR signals are much stronger when acids or amides are irradiated in the presence of l-Phe, showing that they are photosensitized by the aromatic amino acid. The species observed are either alkyl radicals, or radicals formed by abstraction of a hydrogen atom from a molecule of acid or arnide. A mechanism is proposed which involves the capture by carboxyl or amide carbonyl groups of hydrated electrons released by the photo-excited l-Phe, followed by the splitting of the resulting anion free radical with the formation of alkyl radicals, and transfer reactions leading to more stable free radicals. In peptides, which are also photosensitized by l-Phe, electrons are captured preferably by the carboxylic carbonyl groups.

Structure of Phenylalanine Hydroxylase from Colwellia psychrerythraea 34H, a Monomeric Cold Active Enzyme with Local Flexibility around the Active Site and High Overall Stability

The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH(4)) as cofactor and at 25 degrees C (15 degrees C above the optimal growth temperature). Although the enzyme is monomeric with a single L-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S(0.5)](L-Phe) (= 1.1 +/- 0.1 mm) and K(m)(BH(4))(= 0.3 +/- 0.1 mm), as well as high catalytic efficiency at 10 degrees C. However, the half-inactivation and denaturation temperature is only slightly lowered (T(m) approximately 52 degrees C; where T(m) is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH(4) (Gly(87)/Phe(88)/Gly(89)) and l-Phe (Tyr(114)-Pro(118)). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

L-Type amino acids stimulate gastric acid secretion by activation of the calcium-sensing receptor in parietal cells

Parietal cells are the primary acid secretory cells of the stomach. We have previously shown that activation of the calcium-sensing receptor (CaSR) by divalent (Ca(2+)) or trivalent (Gd(3+)) ions stimulates acid production in the absence of secretagogues by increasing H(+),K(+)-ATPase activity. When overexpressed in HEK-293 cells, the CaSR can be allosterically activated by L-amino acids in the presence of physiological concentrations of extracellular Ca(2+) (Ca(o)(2+); 1.5-2.5 mM). To determine whether the endogenously expressed parietal cell CaSR is allosterically activated by L-amino acids, we examined the effect of the amino acids L-phenylalanine (L-Phe), L-tryptophan, and L-leucine on acid secretion. In ex vivo whole stomach preparations, exposure to L-Phe resulted in gastric luminal pH significantly lower than controls. Studies using D-Phe (inactive isomer) failed to elicit a response on gastric pH. H(+)-K(+)-ATPase activity was monitored by measuring the intracellular pH (pH(i)) of individual parietal cells in isolated rat gastric glands and calculating the rate of H(+) extrusion. We demonstrated that increasing Ca(o)(2+) in the absence of secretagogues caused a dose-dependent increase in H(+) extrusion. These effects were amplified by the addition of amino acids at various Ca(o)(2+) concentrations. Blocking the histamine-2 receptor with cimetidine or inhibiting system L-amino acid transport with 2-amino-2-norbornane-carboxylic acid did not affect the rate of H(+) extrusion in the presence of L-Phe. These data support the conclusion that amino acids, in conjunction with a physiological Ca(o)(2+) concentration, can induce acid secretion independent of hormonal stimulation via allosteric activation of the stomach CaSR.

Azide and chloride binding to carboxypeptidase A in the presence of L-phenylalanine

The interaction of chloride with native and cobalt (Co)-substituted carboxypeptidase-A (CPD) has been investigated by 35Cl nuclear magnetic resonance (NMR) spectroscopy in the presence and absence of L-Phe. The affinity constants of azide and chloride toward the Co(II)CPD·L-Phe complex have been measured by electronic spectroscopy. The correlation times determining T 1 and T 2 for the 35Cl nuclei are related to movements inside the cavity. In the presence of L-Phe, the anions bind to the metal with a relatively high affinity at pH values below 6. Anion binding to the Co enzyme can be analyzed in terms of the three protonation state model for the enzyme (EH 2 α EH α E). In the presence of L-Phe, Cl − binds to EH 2 but not to EH or E while N 3 − binds ∼ 34 times tighter to EH 2 than EH, while binding to E is undetectable. The pH-binding constant profile for N 3 − binding to the Co(II)CPD·L-Phe complex is characterized by pK a values of 5.3 and 7.6. The lower value corresponds to the pK a of the group that characterizes the pH- k cat profile for Co(II)CPD while the higher pK a likely reflects the group that characterizes the pH-pK m profile in the alkaline pH region. This group has a pK a of 8.9 in the Co and native enzymes but is shifted to 7.6 in the presence of L-Phe. The present results in conjunction with prior spectral and kinetic studies suggest azide preferentially binds to the enzyme when L-Phe has its amino group protonated. The highly synergistic binding between L-Phe to the active site and azide to the metal may be viewed as caused by replacement of the Glu-270 metal-water hydrogen-bonding network by an electrostatic interaction between ionized Glu-270 and the protonated amino group of L-Phe. Thus, the formation of this E·I species allows ready displacement of the metal bound water by azide.

Altered regulation occurring in the aromatic amino acid biosynthetic pathway of L-tryptophan-producing mutants derived from Bacillus subtilis K.

The regulatory properties of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS) and chorismate mutase (CM) were compared between l-Trp-producing mutants and the wild-type strain of Bacillus subtilis K. The synthesis of DS in the l-Trp producers was elevated 6- to 13-fold compared to that in the wild-type strain, however, its synthesis was still repressed when the mutants were cultured in the presence of l-Phe and/or l-Tyr. The DS from the l-Trp-producers was still sensitive to inhibition by chorismate and prephenate. The specific activity of the CM in the l-Trp- producers was only 3% of that in the case of the wild-type strain, which was due to the loss of activity of the CM 1-type enzyme and which contributed to the derepression of DS and the higher l- Trp accumulation.