Preparation and determination of cytochrome C.

"Experimental Purposes"
1. Learn the chemical and chemical properties of cytochrome C and its biological function.
2. Master the principle of preparing cytochrome C.
3. Master the operation technique of preparing cytochrome C.
C is an
component of the respiratory chain, according to the Experimental Principles. It is an
protein
containing ferrica groups, located between cytochrome b and cytochrome aa3 on the mitochondrial respiratory chain, and the role of cytochrome C is to transfer electrons during biooxidation.
cell pigment C molecule contains high lysine, so isoelectric point bias alkali, pH 10.8, molecular weight of 12000 to 13000 Dalton. It is soluble in water and acidic solution, and more stable, not easy to denature,
tissue
broken, with acidic water solution can be immersed from the cell.
cytochrome C is divided into oxidizing type and also prototype two, because the prototype is more stable and easy to save, generally will be cytochrome C made into a prototype, oxidizing cytochrome C in 408nm, 530nm has the maximum absorption peak, but also prototype cytochrome C maximum absorption peak of 415nm, 520nm and 550nm, this characteristic can be used for cell pigment C content determination.
because cytochrome C is abundant in myocardial tissue

yeast, it is often used as a material for separation preparation. This experiment took pig heart as the material, after acid solution extraction, artificial zeolite adsorption, ammonium sulfate solution was washed away, trinchloro acetate precipitation and other steps to prepare cytochrome C, and determine its content.
the "Experimental Materials"
. 1.
equipment, meat-twisting machine; electromagnetic mixer; electric mixer;
centrifuges
;72l hydrometer; glass column (2.5×30cm); lower bottle;
beaker
(2000, 1000, 500m1); tube; pipe shift; glass
funnel
and gauze; glass rod;
2. Experimental
(1) 2M H2SO4 solution; 1M NH4OH solution; solid ammonium sulfate
(2) 25% ammonium sulfate solution: 100m1 distilled water containing 25 grams of ammonium sulfate, equivalent to a saturation of about 40% at 25 degrees C
(3) 0.2% sodium chloride solution: 0.2 grams of sodium chloride, dissolved with distilled water and fixed to 100 ml.
. (4) BaCl2 reagent: called 12 grams baCl2, dissolved in 100 ml distilled water
(5) 20% tachloro acetate solution
(6) artificial zeolite (60 to 80 eyes)
(7) sodium nihilate (Na2S2O4,2H2O)
experimental operation
1. Preparation of cytochrome C
(1) Material treatment:
take fresh or frozen pig hearts, remove fat and ligaments, wash away blood build-up with water, cut pig hearts into small pieces, and put them in a meat clicing machine.
(2) Extraction:
said to take 500 grams of stranded pig heart muscle, put 2000m1 beaker, add distilled water 1000ml, stirred in an electric mixer with 2M H2S04 pH to 4.0 (at this time the solution is dark purple), stirred at room temperature to extract 2 hours, in the extraction process, so that the pH of the extract remained at about 4.0.
1M NH40H to 6.0 before you finish extracting and stopping stirring. Use eight layers of ordinary gauze to press
filter
and collect filter fluid. The filter residue is added to 750m1 distilled water, and then extracted according to the above conditions for 1 hour, the two extractions are combined.
(3) To reconcile:
with 1M NH4OH to adjust the above extract to pH 7.2 (at this time, isoelectrine close to 7.2 some heteroprotein solubility is small, from the solution precipitated), static 30 to 40 minutes after filtration, the resulting filter is ready to be absorbed through artificial zeolite column.
(4) Adsorption and escape:
artificial zeolite is easy to absorb cytochrome C, after adsorption can be 25% ammonium sulfate washed off, using this characteristic to separate cytochrome C from other hemp proteins. The specific operation is as follows:
(1) artificial zeolite pre-treatment: said to take artificial zeolite 11g, put in a 500m1 beo bowl, add water stirring, with pouring method to remove 12 seconds not to sink fine particles.
(2) Mounting column: select a clean glass column (2.5×30cm) with filter film at the bottom, connect a latex tube at the lower end of the column, clamp it with a clip, add distilled water to the column to 2/3 volume, keep the column vertical, and then fill the column with water, pay attention to the filling of the column once, to avoid air bubbles in the column.
(3) Sample: Once the column is installed, open the clip to release water (a thin layer of water should be retained on the zeolite surface inside the column) and load the prepared extract into the lower bottle so that it can be absorbed by an artificial zeolite column. The speed at which fluid flows out at the lower end of the column is 1.0 ml/min. With the adsorption of cytochrome C, the artificial zeolite in the column gradually changes from white to red, and the outflow should be yellow or slightly red.
(4) Wash: after adsorption, the red artificial zeolite is removed from the column, put into a 500m1 beothing cup, first with tap water, then stirred with distilled water to wash to water clear, and then with 100m1 0.2% NaCl solution wash zeolite three times, and then wash with distilled water to water clear, according to the first column method to reload artificial zeolite into the column.
is washed away with a 25% ammonium sulfate solution at a flow rate of about 2 ml/min, collecting red sequestration containing cytochrome C, which is done when the sulphate red begins to disappear. Artificial zeolite can be used renewablely..