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Streptokinase by Dr.U.Srinivasa, Professor and Head, Srinivas college of pharmacy, Mangalore
This document discusses the production and uses of streptokinase. Streptokinase is an enzyme produced by certain bacteria that activates fibrinolysis, causing the rapid dissolution of blood clots. It is produced through fermentation of streptococcal bacteria and purified using column chromatography and DEAE cellulose. Purified streptokinase is used to treat thromboembolic disorders by lysing blood clots in arteries and veins.
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Streptokinase by Dr.U.Srinivasa, Professor and Head, Srinivas college of pharmacy, Mangalore
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- 2. Introduction: •A blood clot(thrombus) developed in the circulatory system can cause vascular blockage leading to serious consequences including death. • A healthy hemostatic system suppresses the development of blood clots in normal circulation, but reacts extensively in the event of vascular injury to prevent blood loss.
- 3. Outcomes of afailed homeostasis include stroke, pulmonary embolism, deep vain thrombosis and acute myocardial infraction.
- 4. STREPTOCOCCAL FIBRINOLYSIS These enzymeactivates a fibrinolytic enzyme in human serum, which splits fibrin into smaller fragments, thus it causes rapid dissolution of blood clots and fibrinous exudates. Therefore, streptokinase acts indirectly upon a substrate of fibrin or fibrinogen.
- 5. SOURCE This enzyme isproduced by certain bacteria. The most frequently employed for manufacture are: haemolytic streptococci, particularly those of the lancefied groups A, human C and G. Steptodornase is a related enzyme, which act directly upon a substrate of deoxyribonucleoproteins (DNA).
- 6. These are mainconstituents of nuclei. They also present upto 30-70% in purulent exudates. Steptodornase splits them into free purine bases and pyrimidine nucleosides thereby decrese the viscosity of purulent exudates.
- 7. MANUFACTURING PROCESS It isproduced by fermentation process ‘ which involves the following steps:- 1. Preparation of medium 2. Fermentation 3. Purification of the product.
- 8. PREPARATION OF MEDIUM Ingredients:Casein digest solution: It is prepared by dissolving casein in water in specified proportion. It is heated to 100 C and maintained the same temperature, till the solution is clear. The resultant solution is rapidly cooled to 15 C and filtered through a coarse filter paper. Toluene in small quantity is added for the purpose of preservation. It is stored for four days at 20 C and filtered to remove insoluble material.
- 9. 2. Dextrose (acarbohydrate source) 3. Amino acids a)Cysteine in 10%HCl b)Glycine c)Tryptophan. 4. Vitamins a)Thiamine hydrocloride b)Riboflavin c)Nicotinic acid d)Pyridoxine e)Calcium pentothenate a)
- 10. 5. Trace elements– MgSO4, CuSO4, MnSO4, FeSO4 6. Potassium bicarbonate 7. Potassium dihydrogen phosphate 8. Uracil 9. Adenyl sulphate 10. Thioglycolic acid
- 11. FERMENTATION Sterilized medium isinoculated with seed inoculation of bacterium; S. haemolyticus having a bacterial count of 20 billions/ml. fermentation is carried out in a tank for 14hrs at 37C. During this period no pH adjustment, aeration or modification are made.
- 12. Later, dextrose 50%is added and pH is adjusted to 6 at 15 min. interval with 5N sodium hydroxide. After each adjustment of pH, 50% dextrose is added. Fermentation is continued till bacterial count ceases to increase(about 3 hrs).At this stage fermentation medium contains appx. 1000 units/ml
- 13. PURIFICATION Crude streptokinase isfirst dialysed against phosphate buffer then it is applied on modified cellulosic columns and eluted with phosphate buffer with increasing pH and molarity(increasing pH 5.8 to 8.5 and molarity 0.005to 0.1 M).
- 14. pH andmolarity are the important factors in purification. At 0.1 M: streptokinase is eluted > 0.1 M: improper adsorption and separation pH > 8.5:adsorption capacity of cellulose decreases < 5.8:streptokinase is precipitated At pH 8 and molarity 0.75, impurities are eluted.
- 15. PURIFICATION BY DEAE CELLULOSE Crudestreptokinase, phosphate buffer 0.2 M and DEAE cellulose in the proportion of 3:2:1 are stirred for 1 hour and filtered. Cake is washed with phosphate buffer 0.025 m by stirring for 30 mins. It is again filtered and cake is suspended in 0.1 m phosphate buffer by stirring for 1 hour. Filterate is collected which contains pure streptokinase.
- 16. USES Treatment of thromboembolicdisorders for the lysis of pulmonary emboli, arterial thrombus, deep vein thrombus and acute coronary artery thrombosis.