Patent GB-640002-A (GB)
Title: Products derived from blood and methods of obtaining the same
IPC: C12N9/68
Filed: 1947-02-24
Abstract: A profibrinolytic enzyme is obtained from a mammalian blood serum (e.g. bovine), previously freed from prothrombin, by treating the serum with a protein precipitating watersoluble salt, e.g. a sulphate such as ammonium sulphate, at a temperature above freezing for the serum, i.e. above - 5 DEG  C., and below that causing protein denaturation, i.e. below + 10 DEG  C., at the maximum concentration of said salt for precipitating proteins but below that for substantial precipitation of the profibrinolysin contained therein, removing the protein so precipitated and increasing the salt concentration to precipitate the profibrinolysin.  A serum may be freed from prothrombin, thrombin, fibrinogen and fibrin by thawing frozen oxalated blood at room temperatures for 24 hours, precipitating the oxalate by slight excess of calcium chloride, increasing the temperature to 24 DEG  C. and whipping out the fibrin by rapid stirring which is continuedd until all the prothrombin has been converted to thrombin and all the thrombin has been destroyed by anti-thrombin.  Saturated ammonium sulphate solution is added to the serum so prepared and maintained at 4 DEG  to 6 DEG  C., until the serum is 24 to 26 per cent saturated, the precipitated proteins being removed by centrifuging.  The temperature is reduced to -1 DEG  C. to + 1 DEG  C. and saturated ammonium sulphate added to a concentration in the serum of 28 to 31 per cent to precipitate the profibrinolysin, which is separated and washed with ammonium sulphate of 29 per cent saturation.  The profibrinolysin so formed is activated to fibrinolysin by diluting with water, shaking intermittently with chloroform over, say, 30 minutes, separating the aqueous layer, which is dialysed against running water until a precipitate of fibrinolysin forms.  This is purified by dissolving in 0.85 per cent sodium chloride solution, diluting with water, cooling to 0 DEG  C. and adjusting the pH to 5.5 by N.HCl to precipitate the fibrinolysin. Alternatively, a serum free from prothrombin but containing fibrinogen may be obtained by absorbing the prothrombin on magnesium hydroxide, e.g. by stirring a magnesium hydroxide cream into beef plasma and centrifuging to remove the magnesium hydroxide.  Any protein precipitating water-soluble salt such as an alkali metal sulphate may be used instead of ammonium sulphate. Streptococcal activator may also be used for activating the profibrinolysin in place of the chloroform.ALSO:A profibrinolytic product is obtained from mammalian blood serum (particularly bovine) previously freed from prothrombin, by treating the latter with a protein-precipitating water-soluble salt, e.g., a sulphate such as ammonium sulphate at a temperature above freezing for the serum, i.e., above approximately -5 DEG C and below that causing protein denaturation, i.e., below approximately +10 DEG C, at the maximum concentration of said salt for precipitating proteins but below that for substantial precipitation of the profibrinolysin contained therein, removing the protein so precipitated and increasing the salt concentration to precipitate the profibrinolysin.  The serum may be freed from prothrombin, thrombin, fibrinogen and fibrin by thawing frozen oxalated blood for 24 hours at room temperatures, heating to 5 to 8 DEG C by means of a coil containing water at 40 DEG C, precipitating the oxalate by a slight excess of calcium chloride, increasing the temperature to 24 DEG C and whipping out the fibrin by rapid stirring, which is continued until all the prothrombin has been converted to thrombin and all the thrombin has been destroyed by anti-thrombin. Saturated ammonium sulphate solution is added to the serum so prepared and maintained at 4 to 6 DEG C. until the serum is 24 to 26 per cent saturated, the precipitated proteins being removed by centrifuging.  The temperature is reduced to -1 DEG C to +1 DEG C and saturated ammonium sulphate added to 28 to 31 per cent saturated of the serum to precipitate the profibrinolysin, which is separated and washed with 29 per cent saturated ammonium sulphate solution. The profibrinolysin so formed is activated to fibrinolysin by diluting with water, shaking intermittently with chloroform for say 30 minutes, separating the aqueous layer which is dialyzed against running water until a precipitate of fibrinolysin forms.  This is purified by dissolving in 0.85 per cent sodium chloride solution, diluting with water, cooling to 0 DEG C and adjusting the pH to 5.5 with N.HCl, to precipitate the fibrinolysin.  Alternatively, a serum free from prothrombin but containing fibrinogen may be obtained by absorbing the prothrombin in magnesium hydroxide, e.g., by stirring a magnesium hydroxide cream into beef plasma and centrifuging to remove the magnesium hydroxide.  Any protein-precipitating water-soluble salt such as an alkali metal sulphate may be used instead of ammonium sulphate. Streptococcal activator may also be used for activating the profibrinolysin in place of the chloroform.