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ADHP is a fluorogenic substrate for the enzyme Myeloperoxidase (MPO).[1]
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| ln Vitro |
Two distinct techniques were employed to determine compound I's properties, Km and kcat. First, the fluorescence plate reader's syringe function was used to investigate the oxidation of ADHP. H2O2 is dispensed by an autoinjector to start the reaction and provide a series of progress maps. A Km of 31±4 μM and a kcat of 186±6 s 1 were obtained from MPO-mediated ADHP oxidation study. In contrast to the other experiment, where the substrate was maintained constant during the range of 3 to 45 nM MPO, kobs also rose over the experimental range of ADHP doses from 1 to 80 μM. The slope of kobs vs ADHP concentration Kappon yields an apparent second-order rate constant of 2.1±0.2 mM/s[1].
ADHP is oxidized by the MPO-H₂O₂ system to produce the fluorescent product resorufin. Steady-state and transient kinetic analyses were performed to characterize this reaction. For MPO-mediated oxidation of ADHP, the Michaelis constant (Kₘ) was determined to be 31 ± 4 µM with a turnover number (kcat) of 186 ± 6 s⁻¹ using a plate reader method. Stopped-flow fluorescence spectroscopy yielded a Kₘ of 39 ± 11 µM and a kcat of 224 ± 50 s⁻¹. The apparent second-order rate constant (Kₒₙᵃᵖᵖ) for the reaction was 2.1 ± 0.2 mM⁻¹ s⁻¹. ADHP oxidation was used as a reporter system to study and compare the inhibition mechanisms and potencies of various MPO inhibitors (e.g., 4-ABAH, NaN₃).[1] |
| Enzyme Assay |
Steady-State Analysis of MPO Activity: The oxidation of ADHP by MPO was measured as a function of H₂O₂ concentration using a fluorescence plate reader. Reactions containing MPO and ADHP in assay buffer were initiated by the addition of H₂O₂. The fluorescence intensity (excitation 530 nm, emission 590 nm) was monitored over time to determine initial reaction velocities. Kinetic parameters (Kₘ, kcat) for ADHP oxidation by the MPO-H₂O₂ system (Compound I) were derived from plots of initial velocity versus substrate concentration.[1]
Transient State Analysis by Stopped-Flow Kinetics: The kinetics of ADHP oxidation were investigated using a stopped-flow spectrometer. Solutions of MPO and ADHP were rapidly mixed with H₂O₂ in a temperature-controlled apparatus. The time-dependent increase in fluorescence due to resorufin formation was recorded. Progress curves were analyzed to obtain observed rate constants (kₒbₛ) and to perform simultaneous fitting to the integrated Michaelis-Menten equation for more accurate determination of Kₘ and kcat.[1] Fluorescence Data Analysis: Raw fluorescence intensity data from ADHP oxidation assays were converted to molar concentrations of resorufin using a standard curve. Progress curves were fitted to single or double exponential functions to extract kinetic parameters. The relationship between kₒbₛ and the concentration of ADHP or MPO was analyzed to determine the apparent second-order rate constant.[1] Global Analysis of MPO Inhibition Using ADHP as Reporter: Sets of fluorescence progress curves for ADHP oxidation in the presence of various MPO inhibitors (e.g., 4-ABAH, NaN₃) were globally analyzed using DynaFit software. The data were simultaneously fitted to one-step or two-step slow-tight binding inhibition models. This analysis allowed the determination of inhibitory rate constants (k₊₃, k₋₃, k₊₄, k₋₄) and overall inhibition constants (Kᵢ, Kᵢ) for each compound, providing insights into their mechanism of action.[1] |
| References | |
| Additional Infomation |
ADHP (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorescent peroxidase substrate. In the presence of myeloperoxidase (MPO) and H₂O₂, it is oxidized to highly fluorescent halogen (7-hydroxy-3H-phenoxazine-3-one). The proposed mechanism involves the generation of ADHP radicals from the MPO-H₂O₂ complex (compound I), followed by a disproportionation reaction to generate a halogen molecule and a regenerated ADHP molecule. This property makes ADHP a valuable tool for studying the steady-state and rapid kinetics of MPO catalysis and for high-throughput characterization of the relative potency and mechanism of action of MPO inhibitors. The study in this paper was conducted at pH 5.6 and in chloride-free conditions to focus on the peroxidase cycle. [1]
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| Molecular Formula |
C14H11NO4
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|---|---|
| Molecular Weight |
257.24144
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| Exact Mass |
257.068
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| CAS # |
119171-73-2
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| PubChem CID |
167453
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| Appearance |
Light brown to brown solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
618.6±55.0 °C at 760 mmHg
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| Flash Point |
327.9±31.5 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.689
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| LogP |
0.89
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
19
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| Complexity |
335
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
PKYCWFICOKSIHZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H11NO4/c1-8(16)15-11-4-2-9(17)6-13(11)19-14-7-10(18)3-5-12(14)15/h2-7,17-18H,1H3
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| Chemical Name |
1-(3,7-dihydroxyphenoxazin-10-yl)ethanone
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~388.74 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8874 mL | 19.4371 mL | 38.8742 mL | |
| 5 mM | 0.7775 mL | 3.8874 mL | 7.7748 mL | |
| 10 mM | 0.3887 mL | 1.9437 mL | 3.8874 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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