Molecular Docking Studies of Ortho-Substituted Phenols to Tyrosinase Helps Discern If a Molecule Can Be an Enzyme Substrate.

Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more complicated. It can be shown that many of these molecules can become substrates of the enzyme in the presence of catalytic o-diphenol, MBTH, or in the presence of hydrogen peroxide. Docking studies can help discern whether a molecule can behave as a substrate or inhibitor of the enzyme. Specifically, phenols such as thymol, carvacrol, guaiacol, eugenol, isoeugenol, and ferulic acid are substrates of tyrosinase, and docking simulations to the active center of the enzyme predict this since the distance of the peroxide oxygen from the oxy-tyrosinase form to the ortho position of the phenolic hydroxyl is adequate for the electrophilic attack reaction that gives rise to hydroxylation occurring.

Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Considerations about the inhibition of monophenolase and diphenolase activities of tyrosinase. Characterization of the inhibitor concentration which generates 50 % of inhibition, type and inhibition constants. A review.

Tyrosinase is a copper oxidase enzyme which catalyzes the first two steps in the melanogenesis pathway, L-tyrosine to L-dopa conversion and, then, to o-dopaquinone and dopachrome. Hypopigmentation and, above all, hyperpigmentation issues can be originated depending on their activity. This enzyme also promotes the browning of fruits and vegetables. Therefore, control of their activity by regulators is research topic of great relevance. In this work, we consider the use of inhibitors of monophenolase and diphenolase activities of the enzyme in order to accomplish such control. An experimental design and data analysis which allow the accurate calculation of the degree of inhibition of monophenolase activity (iM) and diphenolase activity (iD) are proposed. The IC50 values (amount of inhibitor that causes 50 % inhibition at a fixed substrate concentration) can be calculated for the two activities and from the values of IC50M (monophenolase) and IC50D(diphenolase). Additionally, the strength and type of inhibition can be deduced from these values. The data analysis from these IC50D values allows to obtain the values of [Formula: see text] or [Formula: see text] , or and [Formula: see text] from the values of IC50M. In all cases, the values of the different must satisfy their relationship with IC50M and IC50D.

Interactions of Phenylalanine Derivatives with Human Tyrosinase: Lessons from Experimental and Theoretical tudies.

The pigmentation of the skin, modulated by different actors in melanogenesis, is mainly due to the melanins (protective pigments). In humans, these pigments' precursors are synthetized by an enzyme known as tyrosinase (TyH). The regulation of the enzyme activity by specific modulators (inhibitors or activators) can offer a means to fight hypo- and hyper-pigmentations responsible for medical, psychological and societal handicaps. Herein, we report the investigation of phenylalanine derivatives as TyH modulators. Interacting with the binuclear copper active site of the enzyme, phenylalanine derivatives combine effects induced by combination with known resorcinol inhibitors and natural substrate/intermediate (amino acid part). Computational studies including docking, molecular dynamics and free energy calculations combined with biological activity assays on isolated TyH and in human melanoma MNT-1 cells, and X-ray crystallography analyses with the TyH analogue Tyrp1, provide conclusive evidence of the interactions of phenylalanine derivatives with human tyrosinase. In particular, our findings indicate that an analogue of L-DOPA, namely (S)-3-amino-tyrosine, stands out as an amino phenol derivative with inhibitory properties against TyH.

Fluorometric "AND" logic gate for detection of tyramine and tyrosinase based on in-situ formation of silicon-containing nanoparticles.

BACKGROUND: Tyramine is an important index of food freshness degree, and tyrosinase that can specifically oxidized monophenolamine to catecholamine plays a crucial part in the occurrence and development of melanin-related skin diseases. Therefore, it is crucial to develop sensitive and efficient methods for the detection of tyramine and tyrosinase. RESULTS: In this work, encouraged by tyrosinase-triggered specific oxidation of tyramine to dopamine and the unique fluorescent reaction between dopamine and amino silane, we have developed a one-step synthetic strategy of silicon containing nanoparticles (Si CNPs) for "turn-on" detection of tyramine and tyrosinase. The Si CNPs formed with thoroughly studied mechanism exhibit uniform structure and robust yellow-green fluorescence. The low detection limits for tyramine (1.87 µM) and tyrosinase (0.0029 U/mL) demonstrate admirable sensitivity outstripping most methods. The proposed assay achieves satisfactory results in the determination of tyramine and tyrosinase activity in real samples. Furthermore, we leverage this new fluorescent assay to enable the fabrication of an "AND" Boolean logic gate. SIGNIFICANCE: The entire process can be completed at easily available temperature and pressure with rapid response, convenient operation and visual observation. This fluorescent assay featured with excellent sensitivity, selectivity and stability has considerable prospects in the application of biosensors and disease diagnosis.

A Cu2+ fluorescent chemosensor suitable for quantitative detection of tyrosinase in potatoes over a wide pH range.

Cu2+ as an important trace element plays an essential role in various biologic processes due to the unique redox active nature. For this reason, much effort has been made to develop effective methods for Cu2+ detection. In this study, a novel structure fluorescent chemosensor, 1-(6-(((5-(5, 5-difluoro-1, 3, 7, 9-tetramethyl-5H-4λ4, 5λ4-dipyrrolo[1, 2-c:2', 1'-f][1, 3, 2] diazaborinin-10-yl)quinolin-8-yl)oxy)methyl)pyridin-2-yl)-N, N-bis(pyridin-2-ylmethyl)methanamine (1), was synthesized and characterized by 1H and 13C nuclear magnetic resonance spectroscopy, and electrospray ionization mass spectrometry. Sensor 1 showed an obviously "on-off" fluorescence response to Cu2+ with a 1:1 binding stoichiometry by UV-vis and fluorescence spectrophotometry. The detection limit of sensor 1 to Cu2+ was determined to be 1.9 µM, and the stable pH range for Cu2+ detection was from 3 to 13. Sensor 1 can be used for recognition and detection of tyrosinase in potatoes.

Mushroom Tyrosinase: Six Isoenzymes Catalyzing Distinct Reactions.

"Mushroom tyrosinase" from the common button mushroom is the most frequently used source of tyrosinase activity, both for basic and applied research. Here, the complete tyrosinase family from Agaricus bisporus var. bisporus (abPPO1-6) was cloned from mRNA and expressed heterologously using a single protocol. All six isoenzymes accept a wide range of phenolic and catecholic substrates, but display pronounced differences in their specificity and enzymatic reaction rate. AbPPO3 ignores γ-l-glutaminyl-4-hydroxybenzene (GHB), a natural phenol present in mM concentrations in A. bisporus, while AbPPO4 processes 100 µM GHB at 4-times the rate of the catechol l-DOPA. All six AbPPOs are biochemically distinct enzymes fit for different roles in the fungal life cycle, which challenges the traditional concept of isoenzymes as catalyzing the same physiological reaction and varying only in secondary properties. Transferring this approach to other enzymes and organisms will greatly stimulate both the study of the in vivo function(s) of enzymes and the application of these highly efficient catalysts.

Synthetic Polymer Nanoparticles as an Abiotic Artificial Inhibitor of Tyrosinase.

An innovative methodology is presented for synthesizing synthetic polymer nanoparticles (TINPs) as potent tyrosinase inhibitors. This inhibition strategy combines the integration of two distinct functionalities, phenol, and phenylboronic acid, within the TINPs structure. The phenyl group mimics the natural monophenol substrate, forming a strong coordination with the catalytic copper ion, significantly inhibiting tyrosinase activity. Additionally, phenylboronic acid interacts with catechol, another tyrosinase substrate, further reducing enzyme efficiency. The shared benzene ring in phenyl and phenylboronic acid enhances binding to tyrosinase's hydrophobic pocket near its copper active site, contributing to potent inhibition. TINPs exhibit exceptional performance, boasting an impressive IC50 value of 3.5×10-8 m and an inhibition constant of 9.8×10-9 m. Validation of the approach is unequivocally demonstrated through the successful inhibition of tyrosinase activity and melanin production, substantiated in both in vitro and in vivo scenarios. The mechanism of TINP inhibition is elucidated through circular dichroism and Fourier transform infrared spectroscopy. This study introduces a versatile design approach for developing abiotic polymer-based enzyme inhibitors, expanding possibilities in enzyme inhibition research.

Generation and characterization of retinal pigment epithelium from patient iPSC line to model oculocutaneous albinism (OCA)1A disease.

Oculocutaneous albinism (OCA) is characterized by reduced melanin biosynthesis affecting the retina, thus impairing visual function. The disease pathology of OCA is poorly understood at the cellular level due to unavailability of suitable biological model systems. This study aimed to develop a disease-specific in vitro model for OCA type 1A, the most severe form caused by TYR (tyrosinase) gene mutations, using retinal pigment epithelium (RPE) differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs). A comparative study between healthy and OCA1A RPE cells revealed that while healthy RPE cells exhibited timely onest of pigmentation during differentiation, OCA1A RPE cells failed to pigment even after an extended culture period. This observation was validated by ultrastructural studies using electron microscopy, hinting at melanosome-specific defects. Immunocytochemistry demonstrated abnormal expression patterns of melanogenesis-specific protein markers in OCA1A RPE cells, indicating reduced or absence of melanin synthesis. Next, a quantitative assay was performed to confirm the absence of melanin production in OCA1A RPE cells. Tyrosinase assay showed no activity in OCA1A compared with healthy RPE, suggesting non-functionality of TYR, further corroborated by western blot analysis showing complete absence of the protein. Gene expression by RNA sequencing of healthy and OCA1A RPE cells uncovered differential gene expression associated with lens development, visual perception, transmembrane transporter activity, and key signaling pathways. This disease-in-a-dish model of OCA1A provides an excellent platform to understand disease mechanism, identify potential therapeutic targets, and facilitate gene therapy or gene correction.

Disposable biosensor based on nanodiamond particles, ionic liquid and poly-l-lysine for determination of phenolic compounds.

This study describes the development of a highly sensitive amperometric biosensor for the analysis of phenolic compounds such as catechol. The biosensor architecture is based on the immobilization of tyrosinase (Tyr) on a screen-printed carbon electrode (SPE) modified with nanodiamond particles (ND), 1-butyl-3-methylimidazolium hexafluorophosphate (IL) and poly-l-lysine (PLL). Surface morphologies of the electrodes during the modification process were evaluated by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to investigate the electrochemical characteristics of the modified electrodes. Owing to the synergistic effect of the modification materials, the Tyr/PLL/ND-IL/SPE exhibited high sensitivity (328.2 µA mM-1) towards catechol with a wide linear range (5.0 × 10-8 - 1.2 × 10-5 M) and low detection limit (1.1 × 10-8 M). Furthermore, the method demonstrated good reproducibility and stability. The amperometric response of the biosensor towards other phenolic compounds such as bisphenol A, phenol, p-nitrophenol, m-cresol, p-cresol and o-cresol was also investigated. The analytical applicability of the biosensor was tested by the analysis of catechol in tap water. The results of the tap water analysis showed that the Tyr/PLL/ND-IL/SPE can be used as a practical and effective method for catechol determination.

Methyl 4-pyridyl ketone thiosemicarbazone (4-PT) as an effective and safe inhibitor of mushroom tyrosinase and antibrowning agent.

Enzymatic browning is of concern as it can affect food safety and quality. In this study, an effective and safe tyrosinase inhibitor and anti-browning agent, methyl 4-pyridyl ketone thiosemicarbazone (4-PT), was synthesised and characterised using Fourier-transform infrared (FTIR) spectroscopy, CHNS elemental analysis, and proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR) spectroscopy. The vibrational frequencies of 4-PT were studied theoretically using vibrational energy distribution analysis (VEDA). Density functional theory (DFT) was applied to elucidate its chemical properties, including the Mulliken atomic charges, molecular electrostatic potential (MEP), quantum theory of atoms in molecules (QTAIM) and reduced density gradient non-covalent interactions (RDG-NCIs). Moreover, 4-PT was compared with kojic acid in terms of its effectiveness as a tyrosinase inhibitor and anti-browning agent. The toxicity and physicochemical properties of 4-PT were predicted via ADME evaluation, which proved that 4-PT is safer than kojic acid. Experimentally, 4-PT (IC50 = 5.82 µM, browning index (10 days) = 0.292 ± 0.002) was proven to be an effective tyrosinase inhibitor and anti-browning agent compared to kojic acid (IC50 = 128.17 µM, browning index (10 days) = 0.332 ± 0.002). Furthermore, kinetic analyses indicated that the type of tyrosinase inhibition is a mixed inhibition, with Km and Vmax values of 0.85 mM and 2.78 E-09 µM/s, respectively. Finally, the mechanism of 4-PT for tyrosinase inhibition was proven by 1D, second derivative and 2D IR spectroscopy, molecular docking and molecular dynamic simulation approaches.

Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu2O2 Active Site of oxy-Tyrosinase.

Tyrosinase is a ubiquitous coupled binuclear copper enzyme that activates O2 toward the regioselective monooxygenation of monophenols to catechols via a mechanism that remains only partially defined. Here, we present new mechanistic insights into the initial steps of this monooxygenation reaction by employing a pre-steady-state, stopped-flow kinetics approach that allows for the direct measurement of the monooxygenation rates for a series of para-substituted monophenols by oxy-tyrosinase. The obtained biphasic Hammett plot and the associated solvent kinetic isotope effect values provide direct evidence for an initial H-transfer from the protonated phenolic substrate to the Cu2O2 core of oxy-tyrosinase. The correlation of these experimental results to quantum mechanics/molecular mechanics calculations provides a detailed mechanistic description of this H-transfer step. These new mechanistic insights revise and expand our fundamental understanding of Cu2O2 active sites in biology.

A new efficient multi-stage strategy based on the complementarity of ultrafiltration and high resolution biochromatogram for the screening of skin-whitening candidates from the fibrous root of Bletilla striata.

Ultrafiltration-high performance liquid chromatography (UF-HPLC) and high resolution biochromatogram (HR-biochromatogram), have been proven to be effective methods for the rapid discovery of enzyme inhibitors in natural medicines. In attempt to conquer false-positive and false- negative screening results, a new multi-stage strategy based on the complementarity of UF-HPLC and HR-biochromatogram has been proposed for the fast screening of tyrosinase inhibitory components using the fibrous root of Bletilla striata as a case study. For the first two stages, UF- HPLC and HR-biochromatogram, were applied individually for the screening of high-affinity tyrosinase ligands and tyrosinase inhibitors. After that, the inconsistent results, which yielded two potential active fractions, indicated a third stage screening. Thus, a "strengthen" biochromatogram was established to microfractionate the concentrated extract and further evaluate the tyrosinase inhibitors. The complementarity nature of two different screening methods was firstly explored to distinguish tyrosinase inhibitors from the fibrous root of Bletilla striata. As a result, four compounds were screened, isolated and characterized as new potent tyrosinase inhibitors. The screening results were verified by tyrosinase inhibition assays, melanin inhibitory in zebrafish and molecular docking. All compounds possessed much higher tyrosinase inhibition than α-arbutin, especially, 1-(4- Hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol demonstrated stronger tyrosinase inhibition than kojic acid. This study presented a new screening strategy which had a great potential in rapidly and efficiently exploring tyrosinase inhibitors in complex mixtures. Moreover, it is the first time to reveal the skin-whitening nature of the fibrous root of B. striata, which indicating the promising prospect in the full utilization of B. striata plant.

Immobilization of Agaricus bisporus Polyphenol Oxidase 4 on mesoporous silica: Towards mimicking key enzymatic processes in peat soils.

HYPOTHESIS: The use of immobilized enzyme-type biocatalysts to mimic specific processes in soil can be considered one of the most promising alternatives to overcome the difficulties behind the structural elucidation of riverine humic-derived iron-complexes. Herein, we propose that the immobilization of the functional mushroom tyrosinase, Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4) on mesoporous SBA-15-type silica could contribute to the study of small aquatic humic ligands such as phenols. EXPERIMENTS: The silica support was functionalized with amino-groups in order to investigate the impact of surface charge on the tyrosinase loading efficiency as well as on the catalytic performance of adsorbed AbPPO4. The oxidation of various phenols was catalyzed by the AbPPO4-loaded bioconjugates, yielding high levels of conversion and confirming the retention of enzyme activity after immobilization. The structures of the oxidized products were elucidated by integrating chromatographic and spectroscopic techniques. We also evaluated the stability of the immobilized enzyme over a wide range of pH values, temperatures, storage-times and sequential catalytic cycles. FINDINGS: This is the first report where the latent AbPPO4 is confined within silica mesopores. The improved catalytic performance of the adsorbed AbPPO4 shows the potential use of these silica-based mesoporous biocatalysts for the preparation of a column-type bioreactor for in situ identification of soil samples.

Electro-Biofabrication. Coupling Electrochemical and Biomolecular Methods to Create Functional Bio-Based Hydrogels.

Twenty years ago, this journal published a review entitled "Biofabrication with Chitosan" based on the observations that (i) chitosan could be electrodeposited using low voltage electrical inputs (typically less than 5 V) and (ii) the enzyme tyrosinase could be used to graft proteins (via accessible tyrosine residues) to chitosan. Here, we provide a progress report on the coupling of electronic inputs with advanced biological methods for the fabrication of biopolymer-based hydrogel films. In many cases, the initial observations of chitosan's electrodeposition have been extended and generalized: mechanisms have been established for the electrodeposition of various other biological polymers (proteins and polysaccharides), and electrodeposition has been shown to allow the precise control of the hydrogel's emergent microstructure. In addition, the use of biotechnological methods to confer function has been extended from tyrosinase conjugation to the use of protein engineering to create genetically fused assembly tags (short sequences of accessible amino acid residues) that facilitate the attachment of function-conferring proteins to electrodeposited films using alternative enzymes (e.g., transglutaminase), metal chelation, and electrochemically induced oxidative mechanisms. Over these 20 years, the contributions from numerous groups have also identified exciting opportunities. First, electrochemistry provides unique capabilities to impose chemical and electrical cues that can induce assembly while controlling the emergent microstructure. Second, it is clear that the detailed mechanisms of biopolymer self-assembly (i.e., chitosan gel formation) are far more complex than anticipated, and this provides a rich opportunity both for fundamental inquiry and for the creation of high performance and sustainable material systems. Third, the mild conditions used for electrodeposition allow cells to be co-deposited for the fabrication of living materials. Finally, the applications have been expanded from biosensing and lab-on-a-chip systems to bioelectronic and medical materials. We suggest that electro-biofabrication is poised to emerge as an enabling additive manufacturing method especially suited for life science applications and to bridge communication between our biological and technological worlds.

Covalent immobilization of tyrosinase on magnetic multi-walled carbon nanotubes for inhibitor screening.

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibrating sample magnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13 ± 0.91 µM compared to kojic acid (41.96 ± 0.78 µM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.

High-resolution monophenolase/diphenolase/radical scavenging profiling for rapid screening of natural whitening candidates from Rosa rugosa cv. 'Plena'.

Antioxidants and tyrosinase inhibitory components were successfully screened and separated from Rosa rugosa cv. 'Plena' by high-performance liquid chromatography microfractionation bioactive screening combined with several separation and purification methods. Ethyl acetate extract of Rosa rugosa cv. 'Plena' showed high antioxidant activity and tyrosinase inhibitory activity. High-speed countercurrent chromatography, silica gel column chromatography, and semi-preparative high-performance liquid chromatography were used for the preparative separation of four bioactive components from ethyl acetate extract. Two tyrosinase-inhibiting active substances, flavogallonic acid, and N1 -N5 -N10 -tri-4-p-coumaroylspermidine, were isolated from Rosa rugosa cv. 'Plena', and they showed great monophenolase inhibition activity (half-maximal inhibitory concentration: 664.60 and 23.77 µg/ml, respectively) and excellent diphenolase inhibition activity (half-maximal inhibitory concentration: 23 614.61 and 16.80 µg/ml, respectively). Meanwhile, gallic acid, flavogallonic acid, and ellagic acid were shown to have excellent 1,1-diphenyl-2-picryl-hydrazyl antioxidant activity (half maximal inhibitory concentration: 6.66, 20.17, and 13.45 µg/ml), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) antioxidant activity (half maximal inhibitory concentration: 3.53, 3.83, and 2.78 µg/ml). Molecular docking revealed that flavogallonic acid and N1 -N5 -N10 -tri-4-p-coumaroylspermidine had a strong binding affinity (-9.3 and -10 kcal/mol, respectively) to tyrosinase through hydrogen bonding and hydrophobic interactions.

Preparation, antioxidant and tyrosinase inhibitory activities of chitosan oligosaccharide-hydroxypyridinone conjugates.

Two novel chitosan oligosaccharide (COS)-hydroxypyridone (HPO) conjugates were prepared by reacting chitosan oligosaccharide with 2-chloromethyl-5-hydroxypyridone (HPO), which was synthesized by a series of reactions starting from kojic acid. The degree of substitution of COS-HPO2 reached 1.2, with a yield of 74.9%. The structure of the two conjugates (COS-HPO1 and COS-HPO2) was identified by NMR and FT-IR analysis. The two conjugates showed significantly higher free radical (DPPH•, ABTS+• and •OH) scavenging activity and reducing power than those of COS and HPO (p < 0.05). Both COS-HPO1 and COS-HPO2 possessed significantly stronger tyrosinase inhibitory activity than those of COS, with IC50 values of 0.67 and 0.28 mg/mL for monophenolase, 0.73 and 0.30 mg/mL for diphenolase, respectively. In addition, the conjugates were found to be non-toxic to RAW264.7 macrophages and MRC-5 human lung cells. This work proposes a facile method to enhance the oxidative and tyrosinase inhibitory properties of COS.

Shield, Anchor, and Adhesive Roles of Methylene Blue in Tyrosinase Adsorbed on Carbon Felt for a Flow Injection Amperometric Enzyme Biosensor for Phenolic Substrates and Inhibitors.

Methylene blue (MB) acted as a stabilizer for preventing surface-induced denaturation of tyrosinase (TYR) adsorbed on a carbon felt (CF) surface, which is based on shield and anchor roles preventing the unfavorable conformational change of TYR on the hydrophobic CF surface. Furthermore, MB acted as an effective adhesive for TYR immobilization on CF. The resulting TYR and MB coadsorbed CF (TYR/MB-CF) worked as an excellent working electrode unit in an electrochemical detector in a flow injection amperometric biosensor, which allowed highly sensitive consecutive determination of not only TYR substrates but also competitive inhibitors. Simultaneous adsorption of TYR and MB from their mixed solution was much useful as compared with step-wise separated adsorption of TYR on the MB-adsorbed CF, which suggests that the binding interaction of MB with TYR in the solution phase is important for this phenomenon. Fluorescence and UV-vis spectroscopy revealed that not only electrostatic forces between the cationic MB and anionic amino acid residues of TYR but also hydrophobic interactions via the phenothiazine ring of MB play a principal binding driving force of MB with TYR at the surface of the TYR molecules. Synchronous fluorescence, three-dimensional fluorescence, and circular dichroism (CD) spectroscopy clarified that the conformation and the secondary structure of TYR slightly changed upon the MB binding, implying that MB binding leads to the modification of the original intramolecular bonding in part.

In vitro characterization of the intramelanosomal domain of human recombinant TYRP1 and its oculocutaneous albinism type 3-related mutant variants.

Tyrosinase related protein 1 (TYRP1) is the most abundant melanosomal protein of the melanocyte, where plays an important role in the synthesis of eumelanin, possibly catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid to 5,6-quinone-2-carboxylic acid. Mutations to the TYRP1 gene can result in oculocutaneous albinism type 3 (OCA3), a rare disease characterized by reduced synthesis of melanin in skin, hair, and eyes. To investigate the effect of genetic mutations on the TYRP1 structure, function, and stability, we engineered the intramelanosomal domain of TYRP1 and its mutant variants mimicking either OCA3-related changes, C30R, H215Y, D308N, and R326H or R87G mutant variant, analogous to OCA1-related pathogenic effect in tyrosinase. Proteins were produced in Trichoplusia Ni larvae, then purified, and analyzed by biochemical methods. Data shows that D308N and R326H mutants keep the native conformations and demonstrate no change in their stability and enzymatic activity. In contrast, mutations C30R and R87G localized in the Cys-rich domain show the variants misfolding during the purification process. The H215Y variant disrupts the binding of Zn2+ in the active site and thus reduces the strength of the enzyme/substrate interactions. Our results, consistent with the clinical and in silico studies, show that mutations at the protein surface are expected to have a negligible phenotype change compared to that of TYRP1. For the mutations with severe phenotype changes, which were localized in the Cys-rich domain or the active site, we confirmed a complete or partial protein misfolding as the possible mechanism of protein malfunction caused by OCA3 inherited mutations.