Method for quantitative determination of polyamines
AssigneeTokuyama Soda Kabushiki Kaisha
InventorsMasato Okada, Makoto Sakamoto
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Abstract
A method for the quantitative determination of polyamines, which comprises allowing a polyamine oxidizing enzyme, an .omega.-aminoalkylaldehyde dehydrogenase, an oxidized nicotinamide coenzyme and, as required, an acylpolyamine anidohydrolase to act upon a sample solution containing polyamines (for example, urine, blood and other kinds of body fluid), and measuring the reduced nicotinamide coenzyme thus formed by, for example, colorimetry, thereby determining the amount of said polyamines.
Claims27
A method for the quantitative determination of polyamines, comprising:
- A. allowing a polyamine oxidizing enzyme, an .omega.-aminoalkylaldehyde dehydrogenase and an oxidized nicotinamide coenzyme to act upon a sample of body fluid containing polyamines, and
- B. measuring the reduced nicotinamide coenzyme thus formed, thereby determining the amount of the polyamines.
4-Aminobutanan dehydrogenase having the following properties:
- (1) reactive with 4-aminobutanal in the presence of oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate to form 4-aminobutyric acid and reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate;
- (2) coactive with oxidized nicotinamide adenine dinucleotide and oxidized nicotinamide adenine dinucleotide phosphate as an oxidized-form nicotinamide coenzyme, and upon 4-aminobutanal and 5-aminopentanal as .omega.-aminoalkyladehyde dehydrogenase;
- (3) having an optimum pH in the range of about 7.7 to 8.3;
- (4) retaining more than 90% activity at pH in the range from 4.5 to 8.5 when stored at 5.degree. C. for 24 hours;
- (5) having a molecular weight of 102,000.+-.5,000;
- (6) having two subunits where the molecular weights of the subunits are 50,000.+-.5,000;
- and
- (7) having a specific activity of 120 to 140 units/mg protein.
A process for the production of 4-aminobutanal-dehydrogenase according to claim 22 which comprises cultivating cells of a strain of Micrococcus capable of producing said 4-aminobutanaldehydrogenase in a culture medium containing carbon sources, nitrogen sources and inorganic salts at a temperature in the range of 15.degree. C. to 40.degree. C. and at a pH in the range of from 4.0 to 9.0 and then isloating the 4-aminobutanal dehydrogenase produced from the grown cells.
A colorimetric method for the quantitive determiniation of polyamines in a body fluid comprising:
- A. reacting a sample body fluid with 0.1 t 50 units of polyamine oxidizing enzyme, 0.1 to 50 uinits of an .omega.-aminoalkylaldehyde dehydrogenase and oxidized nicotinamide coenzyme and converting any conjugated polyamines which may be present to free polyamines by using acylpolyamine amidohydrolase;
- B. adding 0.01 to 50 mM of a dye selected from the group consisting of tetrazolium salts, and an electron carrier selected from the group consisting of diaphorase and 1-methoxy-5-methyl phenazinium methyl sulfate in the presence of 0.3% to 10% by weight of a nonionic surfactant at a pH in the range of about 4 to 7;
- and
- C. measuring the absrobance at about 530 nm of the test solution as compared with a standard solution containing a known amount of a polyamine.
Dependencies are inferred from the claim text.