RU2105559C1 - Method of cytochrome c preparing - Google Patents

Abstract

FIELD: biochemistry. SUBSTANCE: method involves salmon heart stuffing extraction with trichloroacetic or sulfuric acid at pH 4.8-5.6, separation of inert proteins on natural adsorbent perlite added to extractive mass at amount 20-30 g/100 g parent raw followed by extract separation, its neutralization to neutral pH value, precipitation of proteins with crystalline ammonium sulfate, cytochrome c precipitation with trichloroacetic acid or sulfuric acid, its washing with ammonium sulfate saturated solution to separate impurities, dissolving in water followed by freezing-thawing, dialysis, sterile filtration and package. The obtained cyrochrome c shows the most similar amino acid composition with that of human cytochrome c. EFFECT: improved method of preparing. 3 cl

Description

 The invention relates to biotechnology.

 A known method of producing cytochrome C (ed. St. N 351546, class A 61 K 35/34) from the hearts of cattle. The method includes grinding the heart into minced meat, extraction of cytochrome C with trichloroacetic acid at a pH of 3.8-4.1; 2 salting out of ballast proteins with crystalline ammonium sulfate, precipitation of cytochrome C with a 20% solution of trichloroacetic acid, separation of the precipitate by centrifugation. The collected precipitate is dissolved in distilled water, frozen, thawed and filtered, then a strong solution of cytochrome C will be left for dialysis against physiological saline for a day. The resulting solution was sterilized, filtered, the filtrate was diluted with saline to a concentration of cytochrome 2.5 mg / ml, sterile filtered, poured into flanges and lyophilized.

 This method does not allow the isolation of cytochrome from the heart of the hydrobint.

 The closest technical solution is a method for producing cytochrome C [1] The method includes grinding the heart of a fur seal into minced meat, extraction of cytochrome C with trichloroacetic acid at pH 3.8 4.1, neutralization with sodium hydroxide to pH 6.0-7.0, salting out of ballast proteins with crystalline ammonium sulfate, precipitation of cytochrome C with a 20% solution of trichloroacetic acid, treatment of the precipitate with a saturated solution of ammonium sulfate, centrifugation, separation of the precipitate by centrifugation. The collected precipitate is dissolved in distilled water, frozen, thawed and filtered, then a strong solution of cytochrome C is put on dialysis against physiological saline for a day. The resulting solution was sterilized, filtered, the filtrate was diluted with saline to a concentration of cytochrome 2.5 mg / mg, sterile filtered, poured into vials and lyophilized.

 The disadvantage of this method is the inability to isolate cytochrome C from the heart of aquatic organisms.

 The problem solved by the invention, obtaining cytochrome C from a new raw material - the hearts of hydrobionts, in which the amino acid composition corresponds to human cytochrome C.

 The essence of the proposed method is as follows.

 As raw materials, the heart of salmon fish species is used. Raw materials are washed from the blood, crushed (homogenized). Stuffing is also washed from the blood. To the minced meat, add a 2.5% solution of trichloroacetic acid (TCA) or 2% sulfuric acid to a pH of 4.8-5.5. Extract for 2 hours with periodic stirring.

 Natural adsorbent perlite was added to the extraction mass in a ratio of 4: 1. The extract was separated by filtration, sodium hydroxide was added to pH 7.0, and the proteins were precipitated with crystalline ammonium sulfate.

To the filtrate was added a 20% solution of TCA or 10% H ₂ SO ₄ . A precipitate formed which was separated by centrifugation. The precipitate is treated with a solution of ammonium sulfate and centrifuged. The precipitate of cytochrome C is dissolved in distilled water and frozen at a temperature of minus 10 15 ^o C, then thawed and filtered. A strong solution of cytochrome C is put on dialysis. The resulting solution was heated and filtered. The filtrate is diluted with physiological saline, sterilely filtered into vials and lyophilized.

 The invention is illustrated by the following examples.

 Example 1

To 300 g of minced salmon hearts (type Oncorhynchus keta) add a 2.5% solution of trichloroacetic acid to a pH of 4.85 at t = 18 ^o C and extracted for two hours with periodic stirring. At the end of the extraction, 60 g (based on 20 g per 100 g of raw material) of treated perlite of the Sil Sil 272 brand are added to the extraction mass. They are aged 30 minutes. The extract is separated on a suction filter. The volume of the extract is 280 ml.

 To neutralize the extract, 10% NaOH is added thereto to a pH of 7.05. Ballast proteins are precipitated with crystalline ammonium sulfate (based on 500 g / l). 100 g of ammonium sulfate are added per 200 ml of extract. Receive 230 ml of the filtrate.

 To the clear filtrate obtained after the first salting out, with stirring, a 20% solution of trichloroacetic acid at a rate of 25 ml / l. A pink precipitate of cytochrome C precipitates. The precipitate is separated by centrifugation at 3000 rpm. Next, the precipitate is washed with a solution of ammonium sulfate at the rate of 500 ml / kg of raw material, centrifuged, the precipitate is separated. The collected precipitate is dissolved in distilled water. The resulting cytochrome solution is frozen. Defrost and filter. A strong solution of cytochrome C is poured into a plastic bag and put on dialysis against saline for a day. The output of cytochrome 0.1 g / kg of feedstock.

 The natural perlite adsorbent is treated as follows: perlite is washed with distilled water to pH 6, then soaked in acidified sulfuric acid to pH 5.8 and kept under stirring for 1 hour. Perlite is squeezed through calico or capron and added to the minced meat.

 Example 2

Take 100 g of minced meat from salmon hearts (type Oncorhynchus nerca) with a shelf life of 4.5 months in the freezer at t = -8 ^o C. Extraction is carried out with 2.5% TCA at pH 5.5 for 2 hours. At the end of extraction, add previously prepared, as in example 1, in an amount of 25 g of Dicalite brand perlite (USA) and kept under stirring for 0.5 hours. 81 ml of extract (pH 5.5) are obtained after filtration on a suction filter.

 10% sodium hydroxide was added to the extract to a pH of 7.3. To precipitate proteins, crystalline ammonium sulfate was added to the extract at a rate of 450 g / l, stirred and incubated for 30 minutes. Filter through paper filters. The volume of the obtained filtrate is 62 ml.

Cytochrome C 10% H ₂ SO ₄ is precipitated from the obtained filtrate, a precipitate of cytochrome C precipitates, is filtered, the supernatant is drained, and the precipitate is treated with a saturated solution of ammonium sulfate and centrifuged. The collected cytochrome C precipitate was dissolved in 5 ml of distilled water. Freeze at t-12 ^o C, thaw and filter to separate cryoprecipitate proteins. A strong solution of cytochrome is placed in a plastic bag, put on dialysis against saline.

 The yield of cytochrome C from the feedstock is 0.090 g / kg of feedstock.

 Example 3

Take 400 g of fresh salmon hearts (a species of Oncorhynchus gorbuscha), washed from the blood, ground into minced meat, washed again from the blood. To the prepared minced meat add 2% H ₂ SO ₄ to a pH of 4.9, t 23 ^o C. Exit to the mode of 20 minutes Extraction is carried out for 2 hours. At the end, the perlite of the brand Flo R (France) treated in Example 1 is added to the extraction mass at the rate of 300 g / kg of raw material, left for 30 minutes to precipitate the proteins. Separate the cake from the extract through calico. Next, neutralize the extract with 10% NaOH to a pH of 7.15.

 Salting out is carried out with crystalline ammonium sulfate at a rate of 450 g / l, 126 g of sulfate is added to 280 ml of the filtrate. After filtration, the filtrate volume is 335 ml.

Precipitated with cytochrome 20% trichloroacetic acid. 6.6 trichloroacetic acid was added to 330 ml of the filtrate at the rate of 20 ml / l. After 20-30 minutes, a precipitate of cytochrome C precipitates. Separate it by centrifugation at 3000 rpm for 10 minutes and treat it with a saturated solution of ammonium sulfate to remove impurities. The collected solution in 1.7 ml of distilled water. The resulting solution of cytochrome C is frozen at -12 ^o C. Thawed and filtered. A strong solution of cytochrome C is poured into a plastic bag and put on dialysis against saline for a day.

 The yield of cytochrome C is 0.070 mg / kg of raw material.

Claims (3)

 1. The method of producing cytochrome C by extraction of the crushed heart muscle of the animal, including the deposition of ballast baskas with crystalline ammonium sulfate, the precipitation of cytochrome C with acid, processing the precipitate with a saturated solution of ammonium sulfate, followed by dissolving the precipitate containing the target product in distilled water, freezing, thawing, filtering and dialysis, characterized in that the salmon heart is used as a feedstock, extraction is carried out at a pH of 4.8-5.5, and at the end of the extraction, extraction Perlite is added to the mixture in a ratio of 20-30 g per 100 g of feedstock.  2. The method according to claim 1, characterized in that the extraction of the crushed cardiac muscle is carried out 2.0 2.5% solution of sulfuric or trichloroacetic acid.  3. The method according to claims 1 and 2, characterized in that the precipitation of cytochrome C is carried out with a 10 20% solution of sulfuric or trichloroacetic acid.