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Cytochrome C synthesis method
- Nov 29, 2018 -

1. Wash the fresh or frozen pig heart, remove the blood clots, fat and tendons, cut into strips, and grind them with a meat grinder. Then, 1.5 times of water was added, and the pH was adjusted to about 4 with 1 mol/L of sulfuric acid, stirred at room temperature, leached for 2 hours, and pressure-filtered. The filtrate was neutralized with 1 mol/L of ammonia to pH = 6.2, and the supernatant was centrifuged. The residue was again extracted once with an equal amount of water, and the two extracts were combined. The extract was adjusted to pH 7.5 with 2 mol/L ammonia water, and the protein was precipitated at rest, centrifuged, and the supernatant was aspirated. Then, 10 g of zeolite was added per liter of the extract, and it was fully adsorbed for 40 minutes under constant stirring, allowed to stand, and the supernatant was discarded. The cytochrome C-adsorbed zeolite was washed 3 times with distilled water, washed 4 times with 0.2% sodium chloride, and washed with distilled water until the washing liquid was clarified. The zeolite was filtered, drained, loaded into a column, and eluted with 25% ammonium sulfate solution to collect the eluent. Solid ammonium sulfate was added to the eluate to a concentration of 45%, and the heteroprotein was completely precipitated and filtered. The solution of trichloroacetic acid (20%) was slowly added to the filtrate, and stirred while stirring. After the cytochrome C precipitate was completely precipitated, the precipitate was collected by centrifugation. The precipitate was dissolved in distilled water and dialyzed against water to a sulfate-free ion. The dialyzed solution was adsorbed through a treated Amberlite IRC-50 (NH+4) resin column, washed with water until clarified, and then mixed with disodium hydrogen phosphate (0.6 mol/L) and sodium chloride (0.4 mol/L). The liquid elutes and the elution rate is slow, and the general flow rate is 2 ml/min. The eluate was dialyzed against distilled water until it was free of chloride ions, and a cytochrome C fine solution was obtained. The yield is 100-150 mg/kg, and the content is more than 20 mg/ml.