Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6456—Plasminogen activators
  • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/814—Enzyme separation or purification
  • Y10S435/815—Enzyme separation or purification by sorption

Definitions

• the present invention relates to a method for extracting a condensed, purified urokinase from human urine on an industrial scale.
• Urokinase is known to be an enzyme occurring in small quantities in the human urine and found effective for treating various forms of thrombosis, because, when intravenously injected into the human body,
• urokinase For the extraction of urokinase from human urine, various available media are known, such as: heavy metals, barium sulfate, silicic acid and its salts, and various ion exchangers.
• the method according to the present invention is characterized by the step of adsorbing the urokinase in human urine on a material never utilized for extraction of urokinase, that is to say, an arcylonitrile synthetic fiber or its fabrics such as Bonnel (trade name of product by Mitsubishi Bonnel), Exlan (trade name of product by Japan Exlan) or Kashimiron (trade name of product by Asahi Kasei).
• a material never utilized for extraction of urokinase that is to say, an arcylonitrile synthetic fiber or its fabrics
• Bonnel trade name of product by Mitsubishi Bonnel
• Exlan trade name of product by Japan Exlan
• Kashimiron trade name of product by Asahi Kasei
• the acrylonitrile synthetic fiber to be used in this invention is so cheap and easily accessible that it can be thrown away after use, and it needs no pretreatment before use. If, however, it is desirable to remove minor stains from the fiber, the purpose is simply attained by preliminarily dipping the fiber in water and stirring it therein. When air bubbles entrapped among fibers are to be eliminated before contacting the fibers with urine, a similar treatment will help.
• Another feature of our new method resides in the fact that manpower for the collection of urine can be saved or even made unnecessary.
• the enormous manpower required for collection of urine can be replaced by the simple collection of small, light acrylonitrile fibers overlying the urinal or filling a portion of the drain pipe connected to the bottom of the urinal.
• the invented method will be highly useful for industrial extraction of urokinase.
• the process according to our new method is roughly as follows: Human urine is brought into contact with acrylonitrile synthetic fibers, so as to adsorb urokinase from the urine into these fibers, which are picked out to be washed with water to remove the residual urine. Elution is then made using an alkaline solution. Since the urine composition is heavily influenced by' the intake of food or water, the movement, time or climate, it is difiicult to define a normal pH value for the urine, but it is supposed to range generally between 4.8 and 7.5 (Hidenobu MAJIMA: Physiology published by Bunkodo, 1968).
• urokinase in this pH range of 4.8-7.5 urokinase can be adsorbed on acrylonitrile synthetic fibers, but for the sake of a higher yield the pH range of 5.5-6.5 would be preferable.
• a 4% aqueous solution of ammonia is employed for the elution of adsorbed urokinase, an elution rate of 94% will be attained.
• the contact between the urine and the acrylonitrile synthetic fibers is rendered more effective by following the above procedure after treating the urine at room temperature with its pH value adjusted to 7.5-9.5 or preferably to 8.5, and thereby removing the resultant sediments.
• the urokinase-containing solution thus obtained is condensed or refined into a preparation suitable for medical purposes. It is well known that, for the purpose of condensation, the following methods are available: salting-o ut by means of ammonium sulfate, sedimentation by means of an organic solvent, Diafio (trade name of product by U.S. Amicon). For the purpose of refining, various ion exchangers may be utilized, but a high-purity urokinase will be obtained by another contact with acrylonitrile synthetic fibers.
• Table 1 gives the yields of urokinase from various fibers when 2 g. of fibers was contacted with 3 l. of urine having a pH value of 6.0. The fibers were washed with water and the unokinase was then eluted using a 4% aqueous solution of ammonia.
• Table 1 demonstrates the superiority of our new method with respect to the yield of urokinase.
• Table 2 is a comparison between conventional methods and our new method.
• EXAMPLE 1 A large volume of fresh urine collected from many healthy people was adjusted to pH 8.5 under agitation, using sodium hydroxide. The resulting sediment was filtered away or eliminated by centrifugal separation. Then, under violent agitation, 6 N hydrochloric acid was gradually added for adjustment of pH 5.5. This was immediately followed by the addition of Bonnel at a rate of 70 g. per 100 l. of urine and by violent agitation. The Bonnel" which had thus been brought into contact with urine was gathered in a large funnel and washed with water until the wash water turned colorless and clear. Then by compression the wash water was removed. Next using 2 l.
• the urokinase adsorbed by the fibers was separated by elution.
• Ammonium sulfate was added to the eluent under agitation to 60% saturation. After agitation had been continued further for one hour, the precipitate was collected for 30 minutes of centrifugal separation at 20,000 G, dissolved in 500 ml. of water, and then subjected to overnight dialysis, using 100 times as much water.
• the solution thus obtained contained about 85% of the urokinase in the original urine, its specific activity being about 4,000 units/mg.
• the solution finally obtained contained about 80% of urokinase in the crude urokinase solution, its specific activity being over 13,000 units/mg.
• EXAMPLE 3 100 g. of kaolin was added to 100 l. of fresh urine from which sediment had been removed by filtration, and then the mixture was thoroughly stirred. The kaolin was collected by centrifugal separation and washed until the washing turned colorless and clear. Next, the kaolin-adsorbed urokinase was separated by elution using 3 l. of a 4% aqueous solution of ammonia and then subjected to overnight dialysis. The crude urokinase solution thus yielded was adjusted to pH 5.5 using 6 N hydrochloric acid. g. of Exlan (trade name of product of Japan Exlan) textile pieces was then added and well agitated. Thereafter the same process as in Example 1 was carried out. Namely, elution was performed using 1 l. of a 4% aqueous solution of ammonia. Thereafter the process was the same as in Example 1.
• the solution thus obtained contained about of urokinase in the crude solution, or about 40% of it in the original urine, its specific activity being over 10,000 units/mg.
• a method for extracting urokinase from a liquid containing said urokinase comprises the steps of bringing a material comprising synthetic acrylonitrile fibers into contact with said liquid until a substantial proportion of said urokinase has been adsorbed by said material, and then separating the adsorbed urokinase from said material by elution.

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• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Enzymes And Modification Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Artificial Filaments (AREA)

Applications Claiming Priority (1)

Publications (1)

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Family Applications (1)

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Cited By (9)

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• 1970
  • 1970-09-04 JP JP45077389A patent/JPS4810232B1/ja active Pending
• 1971
  • 1971-08-23 US US00174204A patent/US3723251A/en not_active Expired - Lifetime
  • 1971-08-31 FR FR7131434A patent/FR2105243B1/fr not_active Expired
  • 1971-08-31 CA CA121,819A patent/CA952457A/en not_active Expired
  • 1971-09-01 GB GB4079771A patent/GB1305510A/en not_active Expired
  • 1971-09-01 DE DE2143816A patent/DE2143816C3/de not_active Expired
  • 1971-09-02 DK DK431571AA patent/DK128568B/da unknown
  • 1971-09-02 NL NL7112068.A patent/NL164091C/xx not_active IP Right Cessation
  • 1971-09-03 BR BR5833/71A patent/BR7105833D0/pt unknown
  • 1971-09-03 SE SE7111204A patent/SE372538B/xx unknown

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