Application of Skyline for Analysis of Protein-Protein Interactions In Vivo.

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein-protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.

Simultaneous optical and electrochemical label-free biosensing with ITO-coated lossy-mode resonance sensor.

In this work we discuss a new label-free biosensing device based on indium tin oxide (ITO) overlaid section of a multimode optical fiber fused silica core. The sensor has been used to optical measurements also simultaneously interrogated electrochemically (EC). Due to optimized thickness and optical properties of ITO film, a lossy-mode resonance (LMR) could be observed in the optical domain, where electrical properties of the film allowed for application of the sensor as a working electrode in an EC setup. It has been confirmed that the LMR response depends on optical properties of the external medium, as well as potential applied to the electrode during cyclic voltammetry. After the ITO surface functionalization with amine groups and covalently attached biotin, the device has been applied for label-free biosensing of avidin in both the domains simultaneously. On the example of biotin-avidin detection system it was demonstrated that when avidin concentration increases a decrease in current and increase in LMR wavelength shift were recorded in EC and optical domain, respectively. Both optical and EC responses follow the protein interaction process, and thus can be used as cross-verification of the readouts. Moreover, an extended information has been achieved comparing to solely EC interrogation, i.e., the grafting process of biotin and avidin was directly monitored optically displaying individual steps of an incubation procedure.

Biotin interference in immunoassays based on biotin-strept(avidin) chemistry: An emerging threat.

Biotinylated antibodies/antigens are currently used in many immunoassay formats in clinical settings for diversified analytes and biomarkers to offer high detection selectivity and sensitivity. Biotin cannot be synthesized by mammals and must be taken as an essential supplement. Normal intake of biotin from various foods and milk causes no effect on the streptavidin/biotin-based immunoassays. However, overconsumption of biotin (daily doses 100-300 mg) poses a significant problem for immunoassays using the biotin-strept(avidin) pair. Biotin interferences are noted in immunoassays of thyroid markers, drugs, hormones, cancer markers, the biomarker for cardiac function (ß-human chorionic gonadotropin), etc. The biotin level required for serious interference in test results varies significantly from test to test and cannot easily be predicted. Immunoassay manufacturers with technologies based on strept(avidin)-biotin binding must investigate the interference from biotin (up to at least 1200 ng/mL or 4.9 µM of biotin) in various formats. There is no concrete solution to circumvent the biotin interference encountered in blood samples, short of biotin removal. Considering the short half-life of biotin in the human body, patients must stop taking biotin supplements for >48 h before the test. However, this scenario is not considered for patients in emergency situations or those with biotinidase deficiency, mitochondrial metabolic disorders or multiple sclerosis. Apparently, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays using strep(avidin)-biotin chemistry. To date, there is no quick and reliable procedure for the detection of biotin at below nanomolar levels in blood and biological samples. Traditional lab-based techniques including HPLC/MS-MS cannot process an enormous number of public samples. Biosensors with high detection sensitivity, miniaturization, low cost, and multiplexing have the potential to address this issue.

A competitive immunoassay for biotin detection using magnetic beads and gold nanoparticle probes.

Haptens are small molecules with low molecular weight that include biotin and many toxins in food. In this study, we used biotin as a model molecule for hapten detection. In this competitive immunoassay anti-biotin antibody-modified magnetic beads (Ab-MBs) and biotinylated thiol-DNA gold nanoparticles (biotin-GNPs) were used. The assay contains three reactions, the mixing of the sample and Ab-MBs, the capture of biotin-GNPs by Ab-MBs and the magnetic attraction. When biotin molecules were absent, the solution was transparent because biotin-GNPs bound to Ab-MBs which were caught by an external magnetic field. When biotin was present, the supernatant was red because the Ab-MBs bound to the analytes and the gold nanoparticles were still in solution. It was possible to complete all the operating steps in 15 min. The limit of detection (LOD) was 2 pmol. This rapid competitive-immunoassay has potential for application in detection of other haptens.

Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin-biotin immunoassays.

Background Biotin interference in streptavidin-based immunoassays causes widespread analytical distortions that may lead to clinical confusion, inappropriate patient management and, ultimately, adverse events. Its prevalence has increased recently due to the increased use of high-dose biotin therapy in specific patient groups (notably multiple sclerosis) and possibly the general community. Methods We have developed a method to deplete biotin from samples using the streptavidin-coated magnetic microparticles that are a component of most susceptible assays. Results We show that high concentrations of spiked biotin can be adequately depleted from serum using this approach, and that gross biochemical derangements can be restored to normality. We also show that biotin in samples derived from multiple sclerosis patients receiving 300 mg biotin daily can be adequately depleted to remove associated analytical interference and restore normal results. The method is applicable to competitive and sandwich immunoassays and importantly, because it does not change the volume of the sample, suitable for the measurement of free thyroid hormone assays. Application of the method does not significantly change the precision of measurement, and for the majority of analytes, the accuracy is not substantially altered. Conclusions Adopting this method enables laboratories to confirm biotin interference in the appropriate clinical setting. Moreover, it enables laboratories to remove the interference and report accurate and reliable results, without the need for patients to withhold beneficial therapy prior to blood tests. Until the biotin tolerance of susceptible assays is improved, our method gives laboratories a safe alternative for reporting results using streptavidin-based methods.

A more sensitive, efficient and ISO 17025 validated Magnetic Capture real time PCR method for the detection of archetypal Toxoplasma gondii strains in meat.

Toxoplasma gondii is a globally prevalent, zoonotic parasite of major importance to public health. Various indirect and direct methods can be used for the diagnosis of toxoplasmosis. Whereas serological tests are useful to prove contact with the parasite has occurred, the actual presence of the parasite in the tissues of a seropositive animal is not demonstrated. For this, a bioassay is still the reference method. As an alternative, various PCR methods have been developed, but due to the limited amount of sample that can be tested, combined with a low tissue cyst density, those have proved to be insufficiently sensitive. A major improvement of the sensitivity was achieved with magnetic capture-based DNA extraction. By combining the hybridization of specific, biotinylated probes with the capture of those probes with streptavidin-coated paramagnetic beads, T. gondii DNA can selectively be "fished out" from a large volume of meat lysate. Still, several studies showed an insufficient sensitivity compared with the mouse bioassay. Here we present a method that is more sensitive (99% limit of detection: 65.4 tachyzoites per 100g of meat), economical and reliable (ISO 17025 validated) by adding a non-competitive PCR inhibition control (co-capture of cellular r18S) and making the release of the target DNA from the streptavidin-coated paramagnetic beads UV-dependent. The presented results demonstrate the potential of the modified Magnetic Capture real time PCR as a full alternative to the mouse bioassay for the screening of various types of tissues and meat, with the additional advantage of being quantitative.

High-dose biotin therapy leading to false biochemical endocrine profiles: validation of a simple method to overcome biotin interference.

BACKGROUND: High-dose biotin therapy is beneficial in progressive multiple sclerosis (MS) and is expected to be adopted by a large number of patients. Biotin therapy leads to analytical interference in many immunoassays that utilize streptavidin-biotin capture techniques, yielding skewed results that can mimic various endocrine disorders. We aimed at exploring this interference, to be able to remove biotin and avoid misleading results. METHODS: We measured free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), parathyroid homrone (PTH), 25-hydroxyvitamin D (25OHD), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, C-peptide, cortisol (Roche Diagnostics assays), biotin and its main metabolites (liquid chromatography tandem mass spectrometry) in 23 plasmas from MS patients and healthy volunteers receiving high-dose biotin, and in 39 biotin-unsupplemented patients, before and after a simple procedure (designated N5) designed to remove biotin by means of streptavidin-coated microparticles. We also assayed fT4, TSH and PTH in the 23 high-biotin plasmas using assays not employing streptavidin-biotin binding. RESULTS: The biotin concentration ranged from 31.7 to 1160 µg/L in the 23 high-biotin plasmas samples. After the N5 protocol, the biotin concentration was below the detection limit in all but two samples (8.3 and 27.6 µg/L). Most hormones results were abnormal, but normalized after N5. All results with the alternative methods were normal except two slight PTH elevations. In the 39 biotin-unsupplemented patients, the N5 protocol did not affect the results for any of the hormones, apart from an 8.4% decrease in PTH. CONCLUSIONS: We confirm that most streptavidin-biotin hormone immunoassays are affected by high biotin concentrations, leading to a risk of misdiagnosis. Our simple neutralization method efficiently suppresses biotin interference.

Magnetic molecularly imprinted polymer for the isolation and detection of biotin and biotinylated biomolecules.

Magnetic separation based on biologically-modified magnetic particles is a preconcentration procedure commonly integrated in magneto actuated platforms for the detection of a huge range of targets. However, the main drawback of this material is the low stability and high cost. In this work, a novel hybrid molecularly-imprinted polymer with magnetic properties is presented with affinity towards biotin and biotinylated biomolecules. During the synthesis of the magneto core-shell particles, biotin was used as a template. The characterization of this material by microscopy techniques including SEM, TEM and confocal microscopy is presented. The application of the magnetic-MIPs for the detection of biotin and biotinylated DNA in magneto-actuated platforms is also described for the first time. The magnetic-MIP showed a significant immobilization capacity of biotinylated molecules, giving rise to a cheaper and a robust method (it is not required to be stored at 4°C) with high binding capacity for the separation and purification under magnetic actuation of a wide range of biotinylated molecules, and their downstream application including determination of their specific targets.

DNA-Encoded Dynamic Combinatorial Chemical Libraries.

Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

Biotin selective polymer nano-films.

BACKGROUND: The interaction between biotin and avidin is utilized in a wide range of assay and diagnostic systems. A robust material capable of binding biotin should offer scope in the development of reusable assay materials and biosensor recognition elements. RESULTS: Biotin-selective thin (3-5 nm) films have been fabricated on hexadecanethiol self assembled monolayer (SAM) coated Au/quartz resonators. The films were prepared based upon a molecular imprinting strategy where N,N'-methylenebisacrylamide and 2-acrylamido-2-methylpropanesulfonic acid were copolymerized and grafted to the SAM-coated surface in the presence of biotin methyl ester using photoinitiation with physisorbed benzophenone. The biotinyl moiety selectivity of the resonators efficiently differentiated biotinylated peptidic or carbohydrate structures from their native counterparts. CONCLUSIONS: Molecularly imprinted ultra thin films can be used for the selective recognition of biotinylated structures in a quartz crystal microbalance sensing platform. These films are stable for periods of at least a month. This strategy should prove of interest for use in other sensing and assay systems.

Expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Synthesis and biological evaluation of biotin conjugates of (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydro-phenanthrene-2,6-dicarbonitrile, an activator of the Keap1/Nrf2/ARE pathway, for the isolation of its protein targets.

The tricycle 1 ((±)-(4bS,8aR,10aS))-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile), a potent activator of the Keap1/Nrf2/ARE pathway, has the potential to be a first in class drug for the treatment of diabetic nephropathy. To identify the protein targets for the development of 1, the (1:1)-diasteromeric mixture of biotinylated tricycles 3a and 3b were designed and synthesized. For the synthesis of 3a and 3b, a new important precursor, hydroxylated tricycle (±)-16 was synthesized from 4 by a C1 α-methyl group oxidation protocol, which involves cyclopalladation of the C1 α-methyl group from a C2-oxime. For the induction of the phase 2 cytoprotective enzyme NQO1 in Hepa1c1c7 murine hepatoma cells, the diasteromeric mixture 3a and 3b shows high potency (CD, 75 nM) although this potency is lower than that of 1 and 16. Thus, biotinylated tricycles 3a and 3b may be promising tools for the isolation of the protein targets of 1.

Determination of biotin in Antarctic krill (Euphausia superba) by high-performance TLC with different post-chromatographic derivatizations.

A new efficient method was developed to detect biotin in Antarctic krill by Vis-absorbance detection. DMF was used after chloroform pretreatment to extract biotin and two chromogenic methods were developed. The development system consisted of dichloromethane/dimethylcarbinol/methanol/glacial acetic acid (3:3:2:0.015, v/v/v/v). Samples were separated on precoated silica gel GF254 high-performance TLC plates. Densitometric analysis of biotin was carried out in the absorbance mode at 400 and 530 nm. The biotin content was determined to be 1.0948 ± 0.0097 and 1.1212 ± 0.0155 mg/g in Antarctic krill with the two chromogenic methods, which had no significant difference.

Diseño de un genosensor electroquímico para la detección indirecta de gluten en alimentos / Electrochemical genosensor for indirect wheat gluten detection in food

Se propone un nuevo genosensor electroquímico para la detección de una secuencia específica de ADN que codifica un fragmento inmunogénico de la Alfa-2-gliadina, proteína del gluten de trigo responsable de la celiaquía. El diseño del genosensor se basa en la formación de una monocapa autoensamblada de sonda de captura y un agente bloqueante, mercaptohexanol, sobre electrodos de oro serigrafiados. Se eligió un ensayo tipo sándwich, utilizando una sonda indicadora marcada con biotina y el conjugado estreptavidina-fosfatasa alcalina como molécula de marcaje. La detección del analito se basó en la medida de la corriente de oxidación del 1-naftol, producto formado por la hidrólisis enzimática del 1-naftil-fosfato, mediante voltamperometría de pulso diferencial. Se investigaron y optimizaron los parámetros implicados en la composición de la fase sensora mediante voltametría cíclica, encontrándose como relación óptima sonda de captura: mercaptohexanol 2 microM:9 mM. Con el objetivo de minimizar las adsorciones inespecíficas, se optimizaron las concentraciones de sonda indicadora y conjugado enzima-estreptavidina, especies involucradas en la fase de medida, obteniéndose como valores óptimos 1 microM y 1,075x10-3 g/L respectivamente. El genosensor propuesto presentó una respuesta lineal entre 20 y 250 nM(AU)

A new electrochemical genosensor has been developed for the detection of a specific DNA sequence that encodes for an immunogenic fragment of Alpha-2-gliadin, protein of gluten wheat that plays an important role in celiac disease. The genosensor is based on a mixed self-assembled monolayer consisting on a capture probe and a diluent molecule, mercaptohexanol, both immobilized on screen-printed gold electrodes. A sandwich-type hybridization assay was selected, using a signaling-DNA probe labeled with biotin and streptavidin-alkaline phosphatase as a reporter molecule. Detection of DNA gluten is based on the measurement of the oxidization current of 1-naphthol, product formed by Alpha-naphthyl phosphate enzymatic hydrolysis, by differential pulse voltammetry. Parameters involved in the sensing phase were investigated and optimized by cyclic voltammetry. The optimal capture probe to mercaptohexanol ratio was found to be 2 micreM:9 mM. In order to minimize unspecific adsorptions, both signaling probe and enzyme-streptavidin conjugate concentrations (measurement phase parameters) were optimized (1 micreM and 1.075·10-3 g/L respectively). A linear response from 20 nM to 250 nM is obtained with the proposed genosensor(AU)

Biotin determination in food supplements by an electrochemical magneto biosensor.

An electrochemical magneto biosensor for the rapid determination of biotin in food samples is reported. The affinity reaction was performed on streptavidin-modified magnetic microbeads as a solid support in a direct competitive format. The biotinylated horseradish peroxidase enzyme (biotin-HRP) competes with free biotin in the sample for the binding sites of streptavidin on the magnetic microbeads. The modified magnetic beads were then easily captured by a magneto graphite-epoxy composite electrode and the electrochemical signal was based on the enzymatic activity of the HRP enzyme under the addition of H(2)O(2) as the substrate and o-phenilendiamine as cosubstrate. The response was electrochemically detected by square wave voltammetry. The limit of detection was 8.4×10(-8) mol L(--1) of biotin (20 µg L(--1)) with a dynamic range from 0.94 to 2.4×10(-7) mol L(--1). Biotin-fortified commercial dietary supplement and infant formula samples were evaluated obtaining good performances in the results. Total time of analysis was 40 min per 20 assays.

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin.

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin.

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.

Inhibition with spontaneous reactivation and the "ongoing inhibition" effect of esterases by biotinylated organophosphorus compounds: S9B as a model.

The biotinylated organophosphorus compound 1-(saligenin cyclic phospho)-9-biotinyldiaminononane (S9B) has been used for the detection, labeling and isolation of the membrane-bound neuropathy target esterase (NTE) as it was considered a specific inhibitor of NTE. After incubation with the soluble fraction of chicken peripheral nerve, most of the soluble esterase activity was highly sensitive to S9B, indicating NTE-like esterases. A kinetic model equation was used to assume a multi-enzymatic system with three different simultaneously occurring molecular phenomena; (1) inhibition; (2) simultaneous spontaneous reactivation; and (3) ongoing inhibition (inhibition during the substrate reaction); to fit the data to analyze kinetic behavior. A high "ongoing inhibition" effect was observed in an enzymatic component. A three-dimensional fit of the model was applied. The best fitting model is compatible with three sensitive enzymatic entities (33, 52 and 15%), and only one spontaneously reactivate. The second-order rate constants of inhibition (k(i)=116 x 10(6), 4.6 x 10(6) and 0.28 x 10(6)M(-1)min(-1), respectively) and the spontaneous reactivation constant for the first sensitive component (k(r)=0.0054 min(-1)) were simultaneously estimated. These parameters are similar to those deduced in spontaneous reactivation experiments of the preinhibited samples with S9B. The estimated proportions of enzymatic components are similar to those previously observed in inhibition experiments with mipafox, demonstrating that this kinetic approach offers consistent results.