RU2033170C1 - Method for production of catalase from erythrocytes of human blood - Google Patents
Method for production of catalase from erythrocytes of human blood Download PDFInfo
- Publication number
- RU2033170C1 RU2033170C1 SU4696379A RU2033170C1 RU 2033170 C1 RU2033170 C1 RU 2033170C1 SU 4696379 A SU4696379 A SU 4696379A RU 2033170 C1 RU2033170 C1 RU 2033170C1
- Authority
- RU
- Russia
- Prior art keywords
- catalase
- red blood
- erythrocytes
- equal volume
- centrifuging
- Prior art date
Links
- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 11
- 102000016938 Catalase Human genes 0.000 title claims abstract description 5
- 108010053835 Catalase Proteins 0.000 title claims abstract description 5
- 210000004369 blood Anatomy 0.000 title claims abstract 3
- 239000008280 blood Substances 0.000 title claims abstract 3
- 238000004519 manufacturing process Methods 0.000 title claims description 3
- 238000000034 method Methods 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 6
- 102000001554 Hemoglobins Human genes 0.000 claims abstract description 4
- 108010054147 Hemoglobins Proteins 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 3
- 239000013049 sediment Substances 0.000 claims abstract 2
- 238000005406 washing Methods 0.000 claims abstract 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 2
- 239000003960 organic solvent Substances 0.000 claims 1
- 239000002504 physiological saline solution Substances 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 238000001556 precipitation Methods 0.000 abstract description 2
- UYJXRRSPUVSSMN-UHFFFAOYSA-P ammonium sulfide Chemical compound [NH4+].[NH4+].[S-2] UYJXRRSPUVSSMN-UHFFFAOYSA-P 0.000 abstract 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 abstract 1
- 230000000087 stabilizing effect Effects 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
Изобретение относится к технологии получения ферментов из сырья животного происхождения, в частности эритроцитов человека, и может быть использовано в фармацевтической промышленности и энзимологии. The invention relates to a technology for producing enzymes from raw materials of animal origin, in particular human red blood cells, and can be used in the pharmaceutical industry and enzymology.
Целью изобретения является интенсификация способа за счет сокращения числа и длительности операций. The aim of the invention is the intensification of the method by reducing the number and duration of operations.
Способ осуществляется следующим способом. The method is carried out in the following way.
5 л эритроцитов донорской крови человека отмывают 0,85-1,0%-ным физиологическим раствором и центрифугируют 15 мин при 2500g, обрабатывают осадок эритроцитов равным объемом дистиллированной воды в течение 4 ч при температуре 4оС. Осаждение гемоглобина проводят обработкой гемолизата эритроцитов смесью пропанола-2 и хлороформа в соотношении 5:3 соответственно при непрерывном помешивании в течение 40-60 мин при температуре -4оС. Затем центрифугируют 15-20 мин при 2500g, удаляют осадок гемоглобина. К полученной надосадочной жидкости (рН 7-7,2) добавляют равный объем дистиллированной воды в соотношении 1: 1, смесь перемешивают в течение 30-40 мин и к ней добавляют сернокислый аммоний из расчета 535-540 г на 1 л экстракта при тщательном перемешивании до полного растворения сернокислого аммония при температуре 25оС.5 l of human blood erythrocytes washed 0.85-1.0% aqueous saline and centrifuged for 15 min at 2500g, the precipitate was treated with erythrocytes with an equal volume of distilled water for 4 hours at 4 ° C. The precipitation of hemoglobin is accomplished by treatment with a mixture of erythrocyte hemolysate 2-propanol and chloroform in a ratio of 5: 3, respectively under stirring for 40-60 min at a temperature of -4 ° C then centrifuged for 15-20 minutes at 2500g, the precipitate was removed by hemoglobin. To the resulting supernatant (pH 7-7.2) add an equal volume of distilled water in a ratio of 1: 1, the mixture is stirred for 30-40 minutes and ammonium sulfate is added to it at a rate of 535-540 g per 1 liter of extract with thorough stirring until complete dissolution of ammonium sulfate at a temperature of 25 about C.
Процесс высаливания целевого продукта с последующим формированием осадка фермента проводят в течение 10-12 ч при 4оС. Осадок фермента отделяют центрифугированием при 2000g в течение 20 мин, растворяют в 50-70 мл дистиллированной воды. Раствор фермента диализуют против дистиллированной воды в течение 24 ч, подвергают стерилизующей фильтрации через мембраны "Миллипор" с диаметром пор 0,2 мкм.Process of salting out the desired product, followed by formation of a precipitate of the enzyme was carried out for 10-12 hours at 4 ° C. The enzyme precipitate was separated by centrifugation at 2000g for 20 min, dissolved in 50-70 ml of distilled water. The enzyme solution is dialyzed against distilled water for 24 hours, sterilized by filtration through Millipore membranes with a pore diameter of 0.2 μm.
Целевой продукт лиофильно высушивают под вакуумом в присутствии стабилизатора сахарозы в концентрации 0,5% в течение 42 ч. The target product is freeze-dried under vacuum in the presence of a sucrose stabilizer in a concentration of 0.5% for 42 hours
Молекулярная масса каталазы 250000 Д, электрофоретическая чистота 98-99,9% удельная специфическая активность 35000-40000 Ед, выход 180-200 мг на 1 л эритроцитов крови. The molecular weight of catalase is 250,000 D, the electrophoretic purity is 98-99.9%, the specific specific activity is 35000-40000 Units, the output is 180-200 mg per 1 liter of red blood cells.
Предложенный способ получения каталазы позволяет сократить число и длительность технологических операций с 8 сут по способу-прототипу до 3 сут 6 ч. The proposed method for producing catalase can reduce the number and duration of technological operations from 8 days by the prototype method to 3 days 6 hours.
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU4696379 RU2033170C1 (en) | 1989-06-07 | 1989-06-07 | Method for production of catalase from erythrocytes of human blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU4696379 RU2033170C1 (en) | 1989-06-07 | 1989-06-07 | Method for production of catalase from erythrocytes of human blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| RU2033170C1 true RU2033170C1 (en) | 1995-04-20 |
Family
ID=21449969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SU4696379 RU2033170C1 (en) | 1989-06-07 | 1989-06-07 | Method for production of catalase from erythrocytes of human blood |
Country Status (1)
| Country | Link |
|---|---|
| RU (1) | RU2033170C1 (en) |
-
1989
- 1989-06-07 RU SU4696379 patent/RU2033170C1/en active
Non-Patent Citations (1)
| Title |
|---|
| Крайнев С.И. О спектральной характеристике каталазы и гемоглобина эритроцитов человека. Биохимия, 1966, т.31, в.1, с.26-32. * |
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