Concentrations Of L-3,4-dihydroxyphenylalanine Research Articles

Tyrosinase from the pulps of local cultivars of Musa spp: Purification, characterization, immobilization, and application in the batch production of l-3,4-dihydroxyphenylalanine.

Tyrosinase, an enzyme involved in browning reactions in plants/crops exposed to mechanical injury, was isolated from the pulp of some different locally available bananas (M. cavendish, M. acuminata, and M. paradisiaca). Tyrosinase from the pulps was extracted, purified, immobilized, and characterized. Thereafter, the potentials of the immobilized tyrosinase in the possible production of l-3,4-dihydroxyphenylalanine (L-DOPA) in an improvised batch reactor was exploited using tyrosine and ascorbate as the substrates. L-DOPA production was monitored via thin-layer chromatography and spectrophotometry (Arnow's method). L-DOPA is a drug that is used in the treatment of Parkinson's disease. Hence, this study exploited a non-chemical route for its synthesis using the tyrosinase obtained from the banana pulps. The purified tyrosinase had an optimum pH and temperature of 6.5 and 7.0, respectively. The molecular weight of the purified tyrosinase was 45 kDa. Quercetin and resorcinol both competitively inhibited the purified tyrosinase from the three cultivars. Immobilized M. cavendish tyrosinase produced the highest concentration (0.60 mM) of L-DOPA after 8 h in an improvised batch reactor. The tyrosinase in the banana pulps serves as a cheap and readily available green route for the possible production of L-DOPA.

Mushroom Tyrosinase Assay is used to Teach Enzyme Kinetics and Inhibition Studies in an Undergraduate Biochemistry Course

A spectrophotometric mushroom tyrosinase assay was optimized for use in the undergraduate biochemistry laboratory course at Virginia Tech. The laboratory exercise is designed to teach students about enzyme kinetics and tyrosinase‐inhibitor interactions. The tyrosinase protein structure was first studied using the python based molecular visualization software PyMOL and a PDB ID from the Protein Data Bank. The PDB ID that was used was PY9W and displays a H(2) L(2) tetramer. The H subunits of the complex contain a copper binding site that is coordinated by 3 histidine residues. The structure/function relationship was further studied using an immersive modeling hypercube. The assay uses white button mushrooms as the source of the tyrosinase enzyme, l‐3,4 dihydroxyphenylalanine (L‐DOPA) as the substrate, and kojic acid as the inhibitor. The optimal amounts of enzyme, substrate, and inhibitor are first determined, then a series of experiments are conducted which test varying concentrations of L‐DOPA against varying concentrations of kojic acid. Change in absorbance is measured at 475 nm in a spectrophotometer and the data is plotted to determine the kinetic constants and type of inhibition. Experimental kinetic constants are then compared to previously reported values. Further protein concentration in the tyrosinase extract is determined using Bradford assays. The incorporation of this exercise into the undergraduate biochemistry laboratory course at Virginia Tech will allow students to explore enzyme kinetics and tyrosinase‐inhibitor interactions.

Carboxylated single-wall carbon nanotubes decorated with SiO2 coated-Nd2O3 nanoparticles as an electrochemical sensor for L-DOPA detection

L-DOPA (L-3,4-dihydroxyphenylalanine), the precursor of dopamine, is widely used in the treatment of Parkinson’s disease, thus determining and monitoring the concentration of L-DOPA is of utmost importance for both medical and scientific purposes. Although many analytical approaches, designed for drug detection and quantification, already exist, there is a constant need for modification of old and tailoring of new, faster, and selective methods. Redox active chemical species, such as L-DOPA, can be measured directly by electrochemical means, whereas electrochemical sensors combine sensitivity and selectivity within a small analytical device. This work demonstrates the development of such electrochemical sensor, based on carboxylated single-wall carbon nanotubes (SWCNT-COOH) decorated with SiO2 coated-Nd2O3 nanoparticles, and further application for the detection of L-DOPA. Developed SWCNT-COOH@Nd2O3-SiO2 sensor shows linear response in the range from 2 µmol L−1 to 52 µmol L−1 analyte concentration, and beside the low detection limit, it is characterized by a fast response time, as well as good life-time, reproducibility and repeatability.

Pea aphid Acyrthosiphon pisum sequesters plant-derived secondary metabolite L-DOPA for wound healing and UVA resistance.

Herbivores can ingest and store plant-synthesized toxic compounds in their bodies, and sequester those compounds for their own benefits. The broad bean, Vicia faba L., contains a high quantity of L-DOPA (L-3,4-dihydroxyphenylalanine), which is toxic to many insects. However, the pea aphid, Acyrthosiphon pisum, can feed on V. faba normally, whereas many other aphid species could not. In this study, we investigated how A. pisum utilizes plant-derived L-DOPA for their own benefit. L-DOPA concentrations in V. faba and A. pisum were analyzed to prove L-DOPA sequestration. L-DOPA toxicity was bioassayed using an artificial diet containing high concentrations of L-DOPA. We found that A. pisum could effectively adapt and store L-DOPA, transmit it from one generation to the next. We also found that L-DOPA sequestration verity differed in different morphs of A. pisum. After analyzing the melanization efficiency in wounds, mortality and deformity of the aphids at different concentrations of L-DOPA under ultraviolet radiation (UVA 365.0 nm for 30 min), we found that A. pisum could enhance L-DOPA assimilation for wound healing and UVA-radiation protection. Therefore, we conclude that A. pisum could acquire L-DOPA and use it to prevent UVA damage. This study reveals a successful co-evolution between A. pisum and V. faba.

Enrichment of Mung Bean with L-DOPA, GABA, Essential Amino Acids via Controlled Biofermentation Strategy

L-DOPA (L-3,4-dihydroxyphenylalanine) the precursor of neurotransmitter dopamine is used in the management of Parkinson disease and effective in controlling diabetic state. Gamma-aminobutyric acid (GABA), a non-protein amino acid is known to have many pharmacological functions and plays a major role in inhibiting neurotransmitter in brain. Whereas essential amino acids can’t synthesize in human body and it must be taken from foods to maintain good immune function. This study aims to evaluate the enrichment of mung bean with L-DOPA, GABA and essential amino acids via controlled solid state fermentation using Rhizopus strain 5351. Fermentation was carried out for a duration up to 48 h at 30 °C and the samples were analyzed at certain time intervals. The concentration of glucosamine and I²-glucosidase, which indicated the growth of fungal was noted low at the early growth stage (0 to 10 h), but it was observing increased linearly within 18 to 48 h growth periods. The L-DOPA was produced after 10 h fermentation time (0.008 g/100 g dry weight, DW) and the highest yield of L-DOPA content (0.07 g/100 g DW) was attained at the fermentation time of 28 h. However, the concentration of L-DOPA was noted decreased after that. The protease activity, free and essential amino acids content also showed a drastic increment within the fermentation period of 10 to 38 h. The highest content of free and essential amino acids (FAAs and EAAs) and the protease activity of fermented mung bean were exhibited at 38 h incubation time, which were 3.74 g/100 g DW, 1.43 g/100 g DW and 18.4 U/g dry weight, respectively. The GABA content of fermented mung bean was found low (0.019 - 0.021 g/100 g DW) at early incubation time (0-10 h), however, it showed a drastic increment in the fermented mung bean after 18 h (0.132 g/100 g DW) and continuously increased until 38 h (0.198 g/100 g DW). This study showed the potential of solid state fermentation as a good strategy to enrich the fermented mung bean with L-DOPA, GABA and other beneficial bioactive compounds which play an important role to maintain good health as it helps to enhance our immune system and regulating neurotransmitter function.

A simple analytical method involving the use of a monolithic silica disk-packed spin column and HPLC-ECD for determination of l-DOPA in plasma of patients with Parkinson's disease

L-DOPA (L-3,4-dihydroxyphenylalanine) is commonly used in the treatment of Parkinson's disease. Monitoring the concentration of L-DOPA in human plasma will enable dose optimization, but is rarely performed because current quantification methods are tedious and time-consuming. In this study, a simple method for the determination of L-DOPA in the plasma of patients with Parkinson's disease was developed. A monolithic silica disk-packed spin column with a phenylboronate moiety, which forms stable anionic complexes with the cis-hydroxyl groups of L-DOPA, was used to extract L-DOPA from plasma with extraction recoveries exceeding 90%. The extracted L-DOPA was then separated on a reversed-phase column and detected electrochemically with a boron-doped diamond electrode. The method, which has a limit of detection of 10 fmol, was then successfully applied for the determination of L-DOPA in the plasma of healthy volunteers and patients with Parkinson's disease.

The effect of L-DOPA on Cryptococcus neoformans growth and gene expression

Cryptococcus neoformans is unusual among melanotic fungi in that it requires an exogenous supply of precursor to synthesize melanin. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. L-3,4-dihydroxyphenylalanine (L-DOPA) is a natural catecholamine that is frequently used to induce melanization in C. neoformans and L-DOPA-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents and heavy metals. Given that C. neoformans needs exogenous substrate for melanization one question in the field is the extent to which melanin-associated phenotypes reflect the presence of melanin or metabolic changes in response to substrates. In this study we analyze the response of C. neoformans to L-DOPA with respect to melanization, gene expression and metabolic incorporation. Increasing the concentration of L-DOPA promotes melanin formation up to concentrations > 1 mM, after which toxicity is apparent as manifested by reduced growth. The timing of C. neoformans cells to melanization is affected by growth phase and cell density. Remarkably, growth of C. neoformans in the presence of L-DOPA results in the induction of relatively few genes, most of which could be related to stress metabolism. We interpret these results to suggest that the biological effects associated with melanization after growth in L-DOPA are largely due to the presence of the pigment. This in turn provides strong support for the view that melanin contributes to virulence directly through its presence in the cell wall.

The Allelochemical L-DOPA Increases Melanin Production and Reduces Reactive Oxygen Species in Soybean Roots

The non-protein amino acid, L-3,4-dihydroxyphenylalanine (L-DOPA), is the main allelochemical released from the roots of velvetbean and affects seed germination and root growth of several plant species. In the work presented here, we evaluated, in soybean roots, the effects of L-DOPA on the following: polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities; superoxide anion (O·-2), hydrogen peroxide (H(2)O(2)), and melanin contents; and lipid peroxidation. To this end, 3-day-old seedlings were cultivated in half-strength Hoagland's solution (pH 6.0), with or without 0.1 to 1.0 mM L-DOPA in a growth chamber (at 25°C, with a light/dark photoperiod of 12/12 hr and a photon flux density of 280 μmol m(-2) s(-1)) for 24 hr. The results showed that L-DOPA increased the PPO activity and, further, the melanin content. The activities of SOD and POD increased, but CAT activity decreased after the chemical exposure. The contents of reactive oxygen species (ROS), such as O·-2 and H(2)O(2), and the levels of lipid peroxidation significantly decreased under all concentrations of L-DOPA tested. These results suggest that L-DOPA was absorbed by the soybean roots and metabolized to melanin. It was concluded that the reduction in the O·-2 and H(2)O(2) contents and lipid peroxidation in soybean roots was due to the enhanced SOD and POD activities and thus a possible antioxidant role of L-DOPA.

L-DOPA neurotoxicity is prevented by neuroprotective effects of erythropoietin

The neurotoxicity of L-3,4-dihydroxyphenylalanine (L-DOPA), one of the most important drugs for the treatment of Parkinson's disease, still remains controversial, although much more data on L-DOPA neurotoxicity have been presented. Considering the well known neuroprotective effects of erythropoietin (EPO), the inhibitory effects of EPO on L-DOPA neurotoxicity need to be evaluated. Neuronally differentiated PC12 (nPC12) cells were treated with different concentrations of L-DOPA and/or EPO for 24h. Cell viability was evaluated using trypan blue, 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining, and cell counting. Free radicals and intracellular signaling protein levels were measured with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. L-DOPA reduced nPC12 cell viability at higher concentrations, but combined treatment with EPO and L-DOPA significantly restored cell viability. Free radicals and hydroxyl radical levels increased by L-DOPA were decreased after combined treatment of L-DOPA and EPO. Levels of survival-related intracellular signaling proteins decreased in nPC12 cells treated with 200μM L-DOPA but increased significantly in cells treated with 200μM L-DOPA and 5μM EPO. However, cleaved caspase-3, a death-related protein, increased in nPC12 cells treated with 200μM L-DOPA but decreased significantly in cells treated with 200μM L-DOPA and 5μM EPO. Pretreatment with LY294002, a phosphatidylinositol 3-kinase inhibitor, prior to combined treatment with EPO and L-DOPA almost completely blocked the protective effects of EPO. These results indicate that EPO can prevent L-DOPA neurotoxicity by activating the PI3K pathway as well as reducing oxidative stress.

Augmentation of postswelling surgical sealant potential of adhesive hydrogels

Two-component hydrogels formed with star polyethylene glycol amine and linear dextran aldehyde polymers (PEG:dextran) show promise as tissue-specific surgical sealants. However, there is a significant loss of adhesion strength to soft tissues following PEG:dextran swelling, which may limit material ability to appose disjoined tissues and prevent leakage from surgical sites. We covalently incorporated the modified amino acid L-3,4-dihydroxyphenylalanine (L-DOPA) into PEG:dextran to enhance postswelling sealant performance. L-DOPA is an essential component of marine animal adhesive plaques and has been used to confer wet adhesion in synthetic materials. As both PEG:dextran cohesion and adhesion are mediated by aldehyde-amine interactions, L-DOPA side-groups make it a potent network modulator with potential to affect multiple material properties. Following 1-h submersion in aqueous media, PEG:dextran doped with 3 mM L-DOPA/M aldehyde on average swelled 50.3% less, had 287.4% greater stiffness, and had 53.6% greater functional adhesion strength compared to the neat hydrogel. Increased concentrations of L-DOPA up to 11 mM L-DOPA/M aldehyde similarly curtailed swelling and mitigated property loss with hydration, but sacrificed initial functional adhesion strength, material modulus, and biocompatibility. Taken together, these data support tailored L-DOPA conjugation as a promising approach to enhance the clinical performance of PEG:dextran sealants.

Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA), one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study, lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of varying concentrations of L-DOPA (1 μM-1 mM). The results indicated a slight augmentation of NO 2 − production at low concentrations of L-DOPA (<100 μM) and complete inhibition of NO 2 − at higher concentrations (500 μM, 1 mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein expression. Total reactive oxygen species (ROS) were detected using 2′, 7′-dichlorofluorescein diacetate fluorescence dye (2′, 7′-DCFC) and there was an increase of intensity with the increasing concentrations of L-DOPA. Furthermore, large amounts of superoxide (O 2 −) and hydrogen peroxide (H 2O 2) were generated from the autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase (SOD); therefore, there was an accumulation of O 2 −, tantamount to elevation in 2′7′-DCFC intensity. Simultaneous accumulation of O 2 − and NO 2 − would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data obtained confirmed that the observed effects on iNOS were not due to the activation of the D 1 or β1 adrenergic receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the increase of O 2 − production in the stimulated glioma cells in vitro.

Nature and kinetic characteristics of L-DOPA uptake in rat renal proximal tubules

The present study was performed with the aim to determine the kinetics and the caracteristics of cellular uptake of L-3,4-dihydroxyphenylalanine (L-DOPA) in rat renal proximal tubules. Incubation of renal tubules at 4°C in the presence of increasing concentrations of L-DOPA results in a linear and concentration-dependent accumulation of the substrate. In experiments carried out at 37°C, the accumulation of L-DOPA in renal tubules was found to be greater than that occurring at 4°C and showed a trend for saturation. The saturable component of L-DOPA uptake was derived from the total amount of L-DOPA accumulated in renal tubules at 37°C subtracted with the values obtained in experiments conducted at 4°C. The Vmax and Km values for the saturable component of L-DOPA uptake in renal tubules were, respectively, 241 ± 32 fmol µg protein(-1)min(-1) and 567 ± 63 µM. Cyanine 863 (5 and 10 µM) was found to decrease the tubular uptake of L-DOPA, whereas probenecid (50 µM) did not change the rate of uptake of L-DOPA into renal tubules. The Vmax and Km values for the saturable component of L-DOPA uptake in renal tubules incubated in the presence of 10 µM cyanine 863 were, respectively, 97 ± 11 fmol µg protein(-1)min(-1) and 160 ± 22 µM. It is suggested that the anionic L-DOPA may behave as an amphoteric substance, both hydroxyl groups in the aromatic ring determining the binding of the molecule to the organic cation transporter.

Simultaneous determination of in vivo hydroxylation of tyrosine and tryptophan in rat striatum by microdialysis-HPLC: relationship between dopamine and serotonin biosynthesis.

Using a microdialysis technique, the rat striatum was perfused with NSD-1015, an inhibitor of aromatic L-amino acid decarboxylase, and the amount of L-3,4-dihydroxyphenylalanine (L-DOPA) and 5-hydroxytryptophan (5-HTP) accumulating in dialysate was measured as an index of in vivo activities of tyrosine hydroxylase and tryptophan hydroxylase. NSD-1015 increased the concentration of L-DOPA much higher than that of 5-HTP in a dose-related manner (1-100 mumol/L). In order to examine the relationship between dopaminergic and serotonergic neurons in the striatum, either 5-HTP or L-DOPA was injected intraperitoneally to rats pretreated with benserazide, an inhibitor of peripheral decarboxylase. 5-HTP administration increased 5-HTP, but decreased L-DOPA in a dose-dependent manner. Conversely, 5-HTP concentration decreased in an association with the increased content of L-DOPA following L-DOPA administration. The decrease of 5-HTP caused by L-DOPA administration was not as remarkable as that of L-DOPA by 5-HTP injection. These results suggest that L-DOPA and 5-HTP, the precursor amino acids for catecholamines and indoleamines, could affect mutually each other neuronal activity through the inhibition of their rate-limiting enzymes.