A Comparison of Mushroom Tyrosinase Dopaquinone and Dopachrome Assays Using Diode-Array Spectrophotometry: Dopachrome Formation vs Ascorbate-Linked Dopaquinone Reduction

Behbahani, Iraj; Miller, Seth; O'Keeffe, David H.

doi:10.1006/mchj.1993.1040

Cited by 30 publications

(18 citation statements)

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“…As the incubation time was increased, the color of the solution turned from light yellow to dark brown (data not shown). As previously reported, the redshift in absorbance and the emergence of dark brown color are clear indications that DOPA are formed in the solution [29]. …”

Section: Resultssupporting

confidence: 60%

Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells

et al. 2017

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The complex network of biochemical and biophysical cues in the pancreatic desmoplasia not only presents challenges to the fundamental understanding of tumor progression, but also hinders the development of therapeutic strategies against pancreatic cancer. Residing in the desmoplasia, pancreatic stellate cells (PSCs) are the major stromal cells affecting the growth and metastasis of pancreatic cancer cells by means of paracrine effects and extracellular matrix protein deposition. PSCs remain in a quiescent/dormant state until they are ‘activated’ by various environmental cues. While the mechanisms of PSC activation are increasingly being described in literature, the influence of matrix stiffness on PSC activation is largely unexplored. To test the hypothesis that matrix stiffness affects myofibroblastic activation of PSCs, we have prepared cell-laden hydrogels capable of being dynamically stiffened through an enzymatic reaction. The stiffening of the microenvironment was created by using a peptide linker with additional tyrosine residues, which were susceptible to tyrosinase-mediated crosslinking. Tyrosinase catalyzes the oxidation of tyrosine into dihydroxyphenylalanine (DOPA), DOPA quinone, and finally into DOPA dimer. The formation of DOPA dimer led to additional crosslinks and thus stiffening the cell-laden hydrogel. In addition to systematically studying the various parameters relevant to the enzymatic reaction and hydrogel stiffening, we also designed experiments to probe the influence of dynamic matrix stiffening on cell fate. Protease-sensitive peptides were used to crosslink hydrogels, whereas integrin-binding ligands (e.g., RGD motif) were immobilized in the network to afford cell-matrix interaction. PSC-laden hydrogels were placed in media containing tyrosinase for 6 hours to achieve in situ gel stiffening. We found that PSCs encapsulated and cultured in a stiffened matrix expressed higher levels of αSMA and hypoxia-inducible factor 1α (HIF-1α), suggestive of a myofibroblastic phenotype. This hydrogel platform offers a facile means of in situ stiffening of cell-laden matrices and should be valuable for probing cell fate process dictated by dynamic matrix stiffness.

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Section: Resultssupporting

confidence: 60%

Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells

et al. 2017

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“…23. Consequently, any data relevant to the determination of reaction product formation must be obtained at least in 2 min [23, 25] as in our experiments.…”

Section: Discussionmentioning

confidence: 99%

Slow‐binding inhibition: A theoretical and practical course for students

Goličnik

Stojan

2004

Biochem Molecular Bio Educ

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Tyrosinase (EC 1.14.18.1) catalyzes the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) to 2,3,5,6-tetrahydro-5,6-dioxo-1H-indole-2-carboxylate (dopachrome), according to the classical Michaelis-Menten kinetic mechanism. The enzyme is strongly but slowly inhibited by ␣-amino-␤-[N-(3-hydroxy-4-pyridone)] propionic acid (L-mimosine), a toxic plant amino acid. Easily available reagents and simple spectrophotometric detection of the product make the experimental characterization and kinetic analysis of tyrosinase action convenient and interesting for teaching purposes. In the present article, we present a theoretical and practical guide to the kinetic analysis of slow-binding inhibition. The effect of L-mimosine on tyrosinase is established by progress curve measurements, carried out on a conventional spectrophotometer equipped with a rapid kinetic accessory. In the analysis, we recommend a classical linearization approach but we also took advantage of a more reliable nonlinear regression method to avoid subjective bias. A multistep procedure starts by careful inspection of the curves to discriminate between candidate mechanisms. Next, the evaluation of initial and steady-state velocities provides information on the enzyme catalytic and Michaelis-Menten constants, as well as the corresponding inhibition constants. Subsequently, an appropriate mathematical derivation enables estimation of the isomerization rate constants characteristic for a slow-binding inhibitor. To conclude, we suggest simultaneous multivariable regression, using all the progress curve data, to cross-check the proposed reaction mechanism and evaluated kinetic constants.

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“…The diphenolase activity of tyrosinase was checked using L-DOPA (Sigma-Aldrich Poland) as substrate (dissolved in the 0.15 mM phosphoric acid to prevent an autoxidation [53] ). The activity assay was performed as described by Behbahani et al [54] with slight modifications. Briefly, all investigated compounds were dissolved in sodium phosphate buffer (50 mM, pH 6.8) to desired test concentrations.…”

Section: Tyrosinase Kinetic Assaymentioning

confidence: 99%

Phosphonic and Phosphinic Acid Derivatives as Novel Tyrosinase Inhibitors: Kinetic Studies and Molecular Docking

Wolińska

Hałdys

Góra

et al. 2019

Chemistry & Biodiversity

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A dozen of phosphonic and phosphinic acid derivatives containing pyridine moiety were synthesized and its inhibitory activity toward mushroom tyrosinase was investigated. Moreover, molecular docking of these compounds to the active site of the enzyme was performed. All the compounds (1–10) demonstrated the inhibitory effect with the IC50 and inhibition constants ranging millimolar concentrations. The obtained results indicate that the compounds show different types of inhibition (competitive, noncompetitive, mixed), but all of them are reversible inhibitors. The obtained outcomes allowed to make the structure–activity relationship (SAR) analysis. Compound 4 ([(benzylamino)(pyridin‐2‐yl)methyl]phenylphosphinic acid) revealed the lowest IC50 value of 0.3 mm and inhibitory constant of Ki 0.076 mm, with noncompetitive type and reversible mechanism of inhibition. According to SAR analysis, introducing bulky phenyl moieties to phosphonic and amino groups plays an important role in the inhibitory potency on activity of mushroom tyrosinase and could be useful in design and development of a new class of potent organophosphorus inhibitors of tyrosinase. Combined results of molecular docking and SAR analysis can be helpful in designing novel tyrosinase inhibitors of desired properties. They may have broad application in food industry and cosmetology.

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A Comparison of Mushroom Tyrosinase Dopaquinone and Dopachrome Assays Using Diode-Array Spectrophotometry: Dopachrome Formation vs Ascorbate-Linked Dopaquinone Reduction

Cited by 30 publications

References 0 publications

Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells

Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells

Slow‐binding inhibition: A theoretical and practical course for students

Phosphonic and Phosphinic Acid Derivatives as Novel Tyrosinase Inhibitors: Kinetic Studies and Molecular Docking

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