A method for screening type I 17β hydroxysteroid dehydrogenase inhibitors using immobilized enzymes
A technology of dehydrogenase inhibitors and immobilized enzymes, applied in the field of screening type I 17β hydroxysteroid dehydrogenase inhibitors, which can solve the difficulties in obtaining raw materials, unstable activity of free aromatase, easy aggregation of free aromatase, etc. question
Examples
Embodiment 1
[0033] Embodiment 1: the preparation of immobilized enzyme
[0034] 1) Extraction of 17β-HSD1
[0035] All steps were carried out on ice at 0-4°C. Remove the chorion and large blood vessels from the placenta, wash it with 50mM PBS (pH=7.4, containing 1% KCl), cut it with scissors, and homogenize it with 50mM PBS (containing 0.25M sucrose). Centrifuge at 2500×g for 30min, discard the precipitate, and centrifuge the supernatant at 60000×g for 60min to precipitate mitochondrial components. Discard the precipitate again, add a certain amount of recrystallized ammonium sulfate solid to the supernatant, the precipitate accumulates, discard the supernatant after centrifugation, and resuspend the precipitate in 50mM PBS (containing 1mM EDTA and 1mM cysteine hydrochloride) , and stored at -80°C.
[0036] 2) Preparation of amino-modified silicon spheres
[0037] Take 200 mg of silica gel with a particle size of 5 μm, add 20 mL of 10% hydrochloric acid, reflux at 90° C. for 12 hour...
example 2
[0040] Example 2: Enzymatic Properties of Immobilized Enzymes
[0041] 1) Optimum reaction conditions for enzymatic reactions
[0042] Take an appropriate amount of immobilized enzyme and free enzyme, add 150 μL of androstenedione with a concentration of 35 μM, and 20 μL of NAPDH with a concentration of 25 mg / mL, and incubate at 37 ° C for 8 h to determine the content of the product testosterone. The optimum reaction pH is 8.0, and the optimum reaction temperature is 40°C. After repeated use 5 times, the immobilized enzyme was inactivated.
[0043] 2) Study on the kinetics of enzymatic reactions
[0044] Take 20 mg of the immobilized enzyme, add 200 μL of NAPDH with a concentration of 25 mg / mL, and 1500 μL of androstenedione with a concentration of 35 μM in sequence. After shaking and mixing, the mixture is evenly divided into 10 tubes. Under the phosphate buffer system of pH=8.0, Incubate at 37°C for 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuge to take the...
example 3
[0046] Example 3: Research of apigenin on immobilized enzyme inhibition
[0047] 1) Apigenin enzymatic inhibition curve
[0048] Take 20 mg of the immobilized enzyme, add 200 μL of NAPDH with a concentration of 25 mg / mL, 1500 μL of androstenedione with a concentration of 35 μM, and 100 μL of apigenin with a concentration of 400 nM in sequence. Under the phosphate buffer system, incubate at 37°C for 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuge to take the supernatant, and use high performance liquid chromatography to determine the content of the product at each time point (deduct corresponding blank), prepare enzymatic curve, calculate enzymatic reaction kinetic coefficient V m and K m .
[0049] 2) Apigenin IC 50 Determination of value
[0050] Take 160 mg of the immobilized enzyme, add 160 μL of NAPDH with a concentration of 25 mg / mL, and 1200 μL of androstenedione with a concentration of 35 μM in sequence, shake and mix well, and divide into 8 tubes, re...
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Abstract
Description
Claims
Application Information
- IPC
- G01N30/02
- Inventors
- 苏梦翔; 周雯迪