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Appropriate Synthesis Essay

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The sulfating activity of the recombinant human SULTS was assayed by using PAP [35S] as sulfate donor. The components of the standard assay mixture to the final volume up to 20µL are 50 mM HEPES buffer at pH 7.0, 1mM DTT, 14 mM PAP [35S], and the substrate. Stock solutions of the substrate prepared in dimethyl sulfoxide and used in enzymatic assay. Then, the substrate is prepared at 10 times the final concentration (1, 10, 50) µM in assay mixture and was added after HEPES buffer and PAP [35S]. The reaction was initiated by the addition of SULT enzyme 2µl.Then, it allowed to complete for 10 minutes at 37C°.After that, the reaction was ended by putting the thin walled tube that contains the essay mixture on heating block source, which is pre-heated to 100 C° .And, this step lasted for 3 minutes. …show more content…
The analysis of [35S] sulfated product using the already prepared TLC procedure with glacial acetic acid: n-butanol [2:1] by volume as solvent system was performed on the supernatant layer. After completing of TLC, TLC plate was air-dried in the hood. Then, it was auto radiographed by using an x-ray film. An autoradiograph shows radioactive spot, which correspond to the sulfated products. The location of sulfated products spots was determined. After that, the cutting from TLC plate and the elusion with 0.5 ml water in glass vial were performed. The counting is done through using liquid scintillation counter where a total of 4.5 ml of Ecolume scintillation liquid was added to each vial and mixed vigorously. In order to examine the pH dependence of sulfation of (S)-O-demethyl naproxen by human SULT1A1, 50mM different buffer are used(sodium acetate at 4.5,5; MES at 5.5,6;HEPES at 7,7.5;TAPS at 8,8.5; CHES at 9,9.5 ; CAPS at

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