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Abram Chernov
Abram Chernov

Magnet Pro 2.3.1



This study introduces a newly developed immobilized DPP-IV superparamagnetic beads for ligand fishing used for the identification of DPP-IV inhibitors in lingonberry extract for the first time. Through this study we also highlight the potential of lingonberry as source of antidiabetic constituents.




Magnet Pro 2.3.1


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Initially, 5 mg of amino-terminated magnetic beads were washed (3 1 mL) with a coupling buffer consisting of a 10 mM phosphate buffer (pH 7.5, 25C). Thereafter, 1 mL of a 5% glutaraldehyde solution was added to the Eppendorf containing the magnetic beads, and the mixture was rotated overnight at room temperature. After that, the beads were washed with coupling buffer (3 1 mL) and 1 mL of a solution of DPP-IV (human recombinant) at 4.0 U mL-1 was added to the magnetic beads and rotated for 48 hours in cold room (at 4C). After magnetic separation, the supernatant was discarded and 1 mL of an endcapping solution (100 mM hydroxylamine, 2.5% NaBH3CN) was added to the magnetic beads. The mixture was kept under rotation for 24h at 4C. Thereafter, the supernatant was discarded, and the DPP-IV linked magnetic beads (BcMag-DPP-IV) were washed (3 1 mL) with assay buffer (50 mM Tris-HCl buffer pH 7.5) and stored in the fridge in the same buffer.


Initially, 100 μL of Tris-HCl buffer and 100 μL of 0.4 mM of GPPN solution was added to a 1.5 mL micro-centrifuge tube containing 0.2 mg of MB-N-(DPP-IV) in 50 μL of assay buffer, and the mixture was incubated at 37 C for 30 minutes. In parallel, a similar experiment was performed with MB-NH2, as a negative control. After incubation, the supernatant was magnetically separated and transferred to a 96-well plate and the absorbance read at 405 nm. The experiment was performed on freshly prepared beads (day 0) and additional four activity measurements were performed using the same bead after storage (4C) for 3, 5, and 9 days. All measurements were performed in triplicates. Normalized enzyme activities were calculated as a ratio of absorbance of the reaction product using MB-N-(DPP-IV) to absorbance of the reaction product using MB-NH2.


A dilution series of six concentrations of DPP-IV (from 0.12 U mL-1 to 0.003 U mL-1) in native state (non-bound) was prepared and 25 μL of each solution was transferred in triplicates to an Eppendorf tube together with 75 μL of 50 mM Tris-HCl buffer (pH 7.5) and 100 μL of a 0.2 mM solution of GPPN. The mixture was left to react for 30 minutes at 37C under gentle shaking. Next, the solutions were transferred to a 96-well microplate; the absorbance of the samples was read at 405 nm and a calibration curve of the absorbances of the reaction product using DPP-IV in its native state was obtained. In parallel, 25 μL of a solution containing 0.05 mg of DPP-IV linked magnetic beads (theoretically equivalent to 10 mU of DPP-IV) was transferred to an Eppendorf tube in triplicate together with 75 μL of 50 mM Tris-HCl buffer (pH 7.5) and 100 μL of a 0.2 mM solution of GPPN. As mentioned above for non-bound DPP-IV, the mixture was left to react for 30 minutes at 37C under gentle shaking. After the reaction time, the beads were magnetically separated, and the solution was transferred to a 96-well microplate and absorbance was read at 405 nm. The absorbance readings of the sample were interpolated in the calibration curve in order to calculate the equivalent activity of the immobilized DPP-IV in relation to DPP-IV in native state.


A model mixture of 5 μM of sitagliptin, diprotin A, hippuric acid and ferulic acid (equimolar) was prepared in buffer 50 mM Tris-HCl, pH 7.5 (S0) and used for the ligand fishing experiment. A volume of 600 μL of the model mixture was added to 1 mg of MB-N-(DPP-IV) and left to incubate for 10 minutes. After magnetic separation, the supernatant (S1) was saved and the magnetic beads were washed with 4 400 μL of assay buffer and wash solutions were saved after magnetic separation (S2-S5). Finally, the magnetic beads were eluted with 3 400 μL of 80% methanol in assay buffer (S6-S8).


A solution of 300 μg mL-1 of the LB extract was prepared in buffer 50 mM Tris-HCl, pH 7.5 (S0) and used for the ligand fishing experiment, in a similar fashion to the model mixture previously described. This LB solution (600 μL) was added to 1 mg of MB-N-(DPP-IV) and left to react for 10 minutes. After magnetic separation, the supernatant (S1) was saved and the magnetic beads were successively washed with 3 400 μL of assay buffer (S2-S4). Finally, the magnetic beads were eluted with 4 400 μL of 80% methanol in assay buffer (S6-S8). The supernatants S0-S8 were analyzed using the same LC-MS instrument as described previously (section 2.4). All experimental parameters, with the exception to the elution gradient, were similar. The following elution gradient was used for analysis of S0-S8 from ligand fishing of LB extract: 0 min, 5% B; 10 min, 25% B; 15 min, 27% B; 30 min, 37% B; 32 min, 50% B; 35 min, 100% B; 40 min, 100%B; 42 min, 5% B.


The catalytic activity of the immobilized DPP-IV magnetic beads was evaluated using the amine functionalized magnetic beads (i.e., MB-NH2) as a control. The assay was performed using the standard substrate Gly-Pro-p-nitroanilide (GPPN) and activity was measured by monitoring production of nitroanilide. The results confirmed that the MB-N-(DPP-IV) beads retained activity and the immobilization of the catalytically active form of DPP-IV (Fig 3). To further investigate potential reusability of the immobilized DPP-IV, activity was measured four times within nine days. The results show that after nine days of storage and four cycles of activity measurement, MB-N-(DPP-IV) only lost 18.8% of its initial activity. Interestingly, the activity appeared to increase at day 3 compared to freshly prepared MB-N-(DPP-IV). While it is common that immobilization increases stability and activity of enzymes the observed increase after three days of storage was peculiar. Overall, these results show that the MB-N-(DPP-IV) are stable at 4C and can maintain catalytic activity through multiple cycles of re-use.


A second ligand fishing experiment was carried out as a proof of concept with the lingonberry extract to identify candidate DPP-IV ligands. The base peak chromatograms from LC-MS analysis (section 2.5) of the crude extract loaded on the magnetic beads (S0), the supernatant after ligand fishing (S1), the washings (S2-S4) and the elutions (S5-S8) are shown in Fig 5. The crude extract (S0) chromatogram showed the presence of seven major peaks and several minor peaks. The overall profile was similar to previous reports [32]. The supernatant (S1) chromatogram of the solution incubated for 10 minutes with MB-N-(DPP-IV) presented a roughly similar profile as S0, which was expected as there will be an excess amount of all analytes for the available binding sites of the immobilized DPP-IV. 041b061a72


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