US3849252A
Expired / Lapsed
US
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Enzyme composition and process for the manufacture thereof
Assignee: DSM Nutritional Products AG
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Filing: 1972-01-14
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Granted: 1974-11-19
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Expires: 1992-01-14
Abstract
Abstract An enzyme composition which has a thrombin-like endopeptidase activity and which is useful in human and veterinary medicine in vivo at low doses as a hemostatic agent or at higher doses as a blood anticoagulant. The enzyme composition is also useful as a reagent for in vitro investigations of the blood of humans or other mammals. The enzyme composition is obtained from the venom of Bothrops atrox or any other snake the venom of which is immunologically cross-reactive with Bothrops atrox venom. The process for preparing the enzyme composition comprises treating an aqueous solution of the snake venom at a pH of about 4 to 6 with phenol or a phenol derivative in order to precipitate an insoluble complex containing the active venom fraction and decomposing the complex in order to release the thrombin-like enzyme composition.
Inventors
- 👤 E Percs
- 👤 K Stocker
- 👤 B Blomback
- 👤 M Blomback
- 👤 B Hessel
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 14:30
CPC Classifications
A; A61; A61L; A61L24/00; A61L24/04; A61L24/10; A61L24/106; A61K; A61K38/00; A61K38/16; A61K38/43; A61K38/46; A61K38/48; A61K38/482; A61K38/4833; C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37; C12Q1/56; A61L2400/00; A61L2400/04; G; G01; G01N; G01N2333/00; G01N2333/435; G01N2333/46; G01N2333/4609; G01N2333/4613; G01N2333/4636; G01N2333/464; G01N2333/745; G01N2333/75; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815; Y10S435/816
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
269 words
AN ENZYME COMPOSITION FROM VENOM OF BOTHROPS ATROX OF ANY OTHER SNAKE THE VENOM OF WHICH IS IMMUNOLOGICALLY CROSS-REACTIVE WITH BOTHROPS ATROX VENOM, THE SAID ENZYME COMPOSITION HAVING, AT LOWER DOSES, A HEMOSTATIC ACTIVITY AND, AT HIGHER DOSES, A BLOOD ANTICOAGULANT ACTIVITY IN HUMANS AND OTHER MAMMALS, AND BEING CHARACTERIZED BY THE FOLLOWING PROPERTIES: A. IT HAS A THROMBIN-LIKE ENDOPEPTIDASE ACTIVITY, B. IT COMPRISES A PEPTIDE COMPONENT AND A CARBOHYDRATE COMPONENT, THE PEPTIDE COMPONENT INCLUDING ONE OR MORE PILYPEPTIDES HAVING A MOLECULAR WEIGHT BETWEEN ABOUT 18,000 AND 55,000 AS DETERMINED BY THE EQUILIBRIUM ULTRACENTRIFUGATION METHOD, C. IT HAS A CARBOHYDRATE (NEUTRAL SUGAR) CONTENT OF ABOUT 2 TO 10 PERCENT BY WEIGHT, BASED ON THE PROTEIN CONTENT, D. IT IS SCARCELY SOLUBLE IN DISTILLED WATER BUT READILY SOLUBLE IN PHYSIOLOGICAL SODIUM CHLORIDE SOLUTION, E. IT FORMS COMPLEXES WITH PEHNOL AND PHENOL DERIVATIVES, WHICH COMPLEXES ARE SCARCELY SOLUBLE OR INSOLUBLE IN WATER, F. IT CAUSES COAGULATION OF FIBRINOGEN BY SELECTIVE ELIMINATION OF FIBRINOPEPTIDES A FROM FIBRIN WITHOUT RELEASING FIBRINOPEPTIDES B, G. IT HAS A THROMBIN-LIKE ACTIVITY CORRESPONDING TO ABOUT 30 TO 450 NH-THROMBIN UNTIS PER MG. OF PROTEIN CONTENT, H. IT SPILTS TOSYLARGININE METHYL ESTER AND GELATIN BUT NOT LYSIN METHYL ESTER, PHENYLALANINE ETHYL ESTER, NACETYLMETHIONINE, N-ACETYLTYROSINE, LEUCYLGLYCINE AND CASEIN, I. IT IS NOT INHIBITED BY HEPARIN, HEPARINOIDS, HIRUDIN, ANTITHROMBINS AND THE POLYVALENT PEPTIDASE INHIBITOR OF KUNITZ, K. IT IS INHIBITED AT AN ENZYME CONCENTRATION OF ABOUT 5 MG. PER ML. TO THE EXTENT OF ABOUT 95 PERCENT WITH RESPECT TO ITS COAGULATION AND ESTERASE ACTIVITY BY 2.5 X 10**3 MOLAR DIISOPROPYL FLUOROPHOSPHATE AT A PH OF 8 WITHIN 3 HOURS,
Claim 2
Dependent Claim
269 words
AN ENZYME COMPOSITION FROM VENOM OF BOTHROPS ATROX OF ANY OTHER SNAKE THE VENOM OF WHICH IS IMMUNOLOGICALLY CROSS-REACTIVE WITH BOTHROPS ATROX VENOM, THE SAID ENZYME COMPOSITION HAVING, AT LOWER DOSES, A HEMOSTATIC ACTIVITY AND, AT HIGHER DOSES, A BLOOD ANTICOAGULANT ACTIVITY IN HUMANS AND OTHER MAMMALS, AND BEING CHARACTERIZED BY THE FOLLOWING PROPERTIES: A. IT HAS A THROMBIN-LIKE ENDOPEPTIDASE ACTIVITY, B. IT COMPRISES A PEPTIDE COMPONENT AND A CARBOHYDRATE COMPONENT, THE PEPTIDE COMPONENT INCLUDING ONE OR MORE PILYPEPTIDES HAVING A MOLECULAR WEIGHT BETWEEN ABOUT 18,000 AND 55,000 AS DETERMINED BY THE EQUILIBRIUM ULTRACENTRIFUGATION METHOD, C. IT HAS A CARBOHYDRATE (NEUTRAL SUGAR) CONTENT OF ABOUT 2 TO 10 PERCENT BY WEIGHT, BASED ON THE PROTEIN CONTENT, D. IT IS SCARCELY SOLUBLE IN DISTILLED WATER BUT READILY SOLUBLE IN PHYSIOLOGICAL SODIUM CHLORIDE SOLUTION, E. IT FORMS COMPLEXES WITH PEHNOL AND PHENOL DERIVATIVES, WHICH COMPLEXES ARE SCARCELY SOLUBLE OR INSOLUBLE IN WATER, F. IT CAUSES COAGULATION OF FIBRINOGEN BY SELECTIVE ELIMINATION OF FIBRINOPEPTIDES A FROM FIBRIN WITHOUT RELEASING FIBRINOPEPTIDES B, G. IT HAS A THROMBIN-LIKE ACTIVITY CORRESPONDING TO ABOUT 30 TO 450 NH-THROMBIN UNTIS PER MG. OF PROTEIN CONTENT, H. IT SPILTS TOSYLARGININE METHYL ESTER AND GELATIN BUT NOT LYSIN METHYL ESTER, PHENYLALANINE ETHYL ESTER, NACETYLMETHIONINE, N-ACETYLTYROSINE, LEUCYLGLYCINE AND CASEIN, I. IT IS NOT INHIBITED BY HEPARIN, HEPARINOIDS, HIRUDIN, ANTITHROMBINS AND THE POLYVALENT PEPTIDASE INHIBITOR OF KUNITZ, K. IT IS INHIBITED AT AN ENZYME CONCENTRATION OF ABOUT 5 MG. PER ML. TO THE EXTENT OF ABOUT 95 PERCENT WITH RESPECT TO ITS COAGULATION AND ESTERASE ACTIVITY BY 2.5 X 10**3 MOLAR DIISOPROPYL FLUOROPHOSPHATE AT A PH OF 8 WITHIN 3 HOURS,
Claim 3
Dependent Claim
128 words
IT IS COMPETITIVELY INHIBITED BY TOSYLARGININE METHYL ESTER AND COMPLETELY INHIBITED BY THE ANTIBODY CONTAINED IN ANTIBOTHROPS SERUM, M. IT IS NOT FIXED AND, THEREFORE, NOT INACTIVATED BY FIBRIN. N. IT HAS A SUBSTANTIALLY LONGER BIOLOGICAL HALFLIFE TIME THAN HEPARIN, O. IT CAUSES THE FORMATION OF A FIBRIN DERIVATIVE WHICH INTERACTS WITH EXCESS FIBRINOGEN TO FORM A NONPOLYMERIZABLE FIBRIN-FIBRINOGEN COMPLEX, P. IT ACTS ON FIBRINOGEN WITH FORMATION OF A FIBRIN WHICH HAS PHYSICO-CHEMICAL PROPERTIES DIFFERENT FROM THOSE OF FIBRIN PRODUCED BY THROMBIN INSOFAR AS IT IS RAPIDLY HYDROLYZED BY FIBRINOLYTIC ENZYMES EVEN IN THE PRESENCE OF ACTIVATED FACTOR XIII AMD OF CALCIUM IONS AND IS SOLUBLE IN 5-MOLAR AQUEOUS UREA, AND Q. IT CAUSE IN VIVO A DEFIBRINOGENATION AND A SECONDARY FIBRINOLYSIS WITHOUT HEMORRHAGIC AND THROMBOEMBOLIC SIDE EFFECTS.
Claim 4
Dependent Claim
128 words
IT IS COMPETITIVELY INHIBITED BY TOSYLARGININE METHYL ESTER AND COMPLETELY INHIBITED BY THE ANTIBODY CONTAINED IN ANTIBOTHROPS SERUM, M. IT IS NOT FIXED AND, THEREFORE, NOT INACTIVATED BY FIBRIN. N. IT HAS A SUBSTANTIALLY LONGER BIOLOGICAL HALFLIFE TIME THAN HEPARIN, O. IT CAUSES THE FORMATION OF A FIBRIN DERIVATIVE WHICH INTERACTS WITH EXCESS FIBRINOGEN TO FORM A NONPOLYMERIZABLE FIBRIN-FIBRINOGEN COMPLEX, P. IT ACTS ON FIBRINOGEN WITH FORMATION OF A FIBRIN WHICH HAS PHYSICO-CHEMICAL PROPERTIES DIFFERENT FROM THOSE OF FIBRIN PRODUCED BY THROMBIN INSOFAR AS IT IS RAPIDLY HYDROLYZED BY FIBRINOLYTIC ENZYMES EVEN IN THE PRESENCE OF ACTIVATED FACTOR XIII AMD OF CALCIUM IONS AND IS SOLUBLE IN 5-MOLAR AQUEOUS UREA, AND Q. IT CAUSE IN VIVO A DEFIBRINOGENATION AND A SECONDARY FIBRINOLYSIS WITHOUT HEMORRHAGIC AND THROMBOEMBOLIC SIDE EFFECTS.
Claim 5
Dependent Claim
32 words
The enzyme composition according to claim 1 which, after having been subjected to hydrolysis at 110*C. for 22 to 72 hours with constant boiling hydrochloric acid, shows the following amino acid analysis:
Claim 6
Dependent Claim
171 words
either a. subjecting the said isotonic solution to a fractional salt precipitation in order to first eliminate ballast substances and then to precipitate a fraction containing the enzyme material, dissolving the said fraction in distilled water, adjusting the solution to a pH of about 3 to 4, heating the solution for a few minutes to about 2 hours at temperatures of about 30-50*C., separating precipitated proteins and adjusting the pH of the residual solution to about 4 to 6, or b. adjusting the said isotonic solution to a pH of 3 to 4 and heating the same for a few hours to about 2 hours at temperatures of about 30*-50*C., separating precipitated proteins, adjusting the residual solution to a pH of about 4.5 to 7, and subjecting the same to a fractional salt precipitation in order to first eliminate ballast substances and then precipitate a fraction containing the active enzyme material, dissolving the said fraction in distilled water and adjusting the solution to a pH of about 4 to 6, and
Claim 7
Dependent Claim
171 words
either a. subjecting the said isotonic solution to a fractional salt precipitation in order to first eliminate ballast substances and then to precipitate a fraction containing the enzyme material, dissolving the said fraction in distilled water, adjusting the solution to a pH of about 3 to 4, heating the solution for a few minutes to about 2 hours at temperatures of about 30-50*C., separating precipitated proteins and adjusting the pH of the residual solution to about 4 to 6, or b. adjusting the said isotonic solution to a pH of 3 to 4 and heating the same for a few hours to about 2 hours at temperatures of about 30*-50*C., separating precipitated proteins, adjusting the residual solution to a pH of about 4.5 to 7, and subjecting the same to a fractional salt precipitation in order to first eliminate ballast substances and then precipitate a fraction containing the active enzyme material, dissolving the said fraction in distilled water and adjusting the solution to a pH of about 4 to 6, and
Claim 8
Dependent Claim
20 words
The enzyme composition according to claim 1 which is free from neurotoxin, bradykinin-forming proteases, phospholipases, amino acid oxydases and phosphatases.
Claim 9
Dependent Claim
90 words
adding phenol or a phenol derivative to the solution obtained according to (a) or (b) in order to precipitate an insoluble complex containing the enzyme material, and either c. decomposing the complex by treatment with dilute acetic acid in a polar organic solvent and isolating the released, undissolved enzyme material, or d. treating the complex in an aqueous medium with a base at a pH of about 7.5 to 8.5 and isolating the released enzyme material by ultrafiltration, dialysis or gel filtration, and further purifying the thus obtained enzyme material.
Claim 10
Dependent Claim
90 words
adding phenol or a phenol derivative to the solution obtained according to (a) or (b) in order to precipitate an insoluble complex containing the enzyme material, and either c. decomposing the complex by treatment with dilute acetic acid in a polar organic solvent and isolating the released, undissolved enzyme material, or d. treating the complex in an aqueous medium with a base at a pH of about 7.5 to 8.5 and isolating the released enzyme material by ultrafiltration, dialysis or gel filtration, and further purifying the thus obtained enzyme material.
Claim 11
Dependent Claim
37 words
The enzyme composition according to claim 1 which, in highly purified form, has the following amino acid sequence starting from the N-terminal chain end: valine -> isoleucine -> glycine -> glycine -> aspartic acid -> glutamic acid.
Claim 12
Dependent Claim
10 words
Process for the preparation of an enzyme composition which comprises
Claim 13
Dependent Claim
10 words
Process for the preparation of an enzyme composition which comprises
Claim 14
Dependent Claim
23 words
The process according to claim 5 wherein the purification of the enzyme composition is carried out by chromatography on a basic ion exchanger.
Claim 15
Dependent Claim
29 words
The process according to claim 5 wherein the said fractional salt precipitations are carried out by means of ammonium, alkali metal or alkaline earth metal salts of mineral acids.
Claim 16
Dependent Claim
31 words
The process according to claims 5 wherein the fractional salt precipitations are carried out by means of ammonium sulphate at a pH of about 4.5 to 7, preferably at about 6.5.
Claim 17
Dependent Claim
39 words
The process according to claim 5 wherein phenol carboxylic acids, alkyl phenols, halogenated phenols, halogenated or alkylated phenol carboxylic acids or salts of phenol carboxylic acids or derivatives thereof are used as phenol derivatives for forming the insoluble complex.
Claim 18
Dependent Claim
19 words
The process according to claims 5 wherein phenol, cresol or sodium salicylate is used for forming the insoluble complex.
Claim 19
Dependent Claim
50 words
The process according to claim 5 whereIn the purification of the enzyme composition is carried out by chromatography on gels such as polydextran, polyacrylamide, agarose or agar gels using mixtures of aliphatic carboxylic acids with water or mixtures of aqueous buffers with water-miscible organic solvents as flowing agent or eluant.
Claim 20
Dependent Claim
31 words
The process according to claim 1 wherein the fractional salt precipitations are carried out by means of ammonium sulphate at a pH of about 4.5 to 7, preferably at about 6.5.
Claim 21
Dependent Claim
19 words
The process according to claim 9 wherein phenol, cresol or sodium salicylate is used for forming the insoluble complex.
References Cited (Backward Citations)
US3657416A
Cited
US3711376A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1977-11-18 | 1978-05-20 | 1978-11-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1981-11-18 | 1982-05-20 | 1982-11-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1985-11-18 | 1986-05-20 | 1986-11-19 | $7700.00 | Window Closed / Lapsed |