Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving glucose or galactose
Patent
1995-11-29
1997-12-23
Leary, Louise
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving glucose or galactose
435 4, 435 18, 435 15, 436 63, 436 74, 436 79, C12Q 154, C12Q 100, C12Q 134, C12Q 148
Patent
active
057006520
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a method for quantitatively determining sodium ions using .beta.-galactosidase in the presence of a cation which competes with the sodium ion.
BACKGROUND ART
As a method for chemically determining the amount of sodium ions in a biosample, there is known a method utilizing a .beta.-galactosidase reaction which increases enzyme activity in proportion to the amount of sodium ions. In this method, Cryptfix.TM. 221 is used to prevent the Chemistry, 34:2295 (1988)!. There is also disclosed a method wherein lithium ion is used instead of Cryptfix.TM. 221 in the above-mentioned determination method, but no concrete example is disclosed which uses lithium ion alone (Japanese Unexamined Patent Publication/PCT No. 1-503596) (Dec. 7, 1989).
A method using a bicyclic crown ether such as Cryptfix.TM. 221 has the following character. Since the dissociation rate of the cryptate is small, a long time is needed for the prereaction before the start of measurement. Therefore, the operation of the method is not simple. In addition, since Cryptfix.TM. 221 is preincubated with .beta.-galactosidase because of the above-mentioned reasons, the stability of .beta.-galactosidase is damaged by Cryptfix.TM. 221 and it becomes impossible to quantitatively determine sodium ions at a low concentration. Furthermore, the pH of the reaction solution is restricted to the alkaline area. In view of these problems, the development of a better method for quantitative determination of sodium ions is desired.
DISCLOSURE OF THE INVENTION
The present invention relates to a method for quantitatively determining sodium ions in a sample using .beta.-galactosidase in an aqueous medium, which method is characterized in that a .beta.-galactosidase reaction is carried out in the presence of a cation which competes with the sodium ion.
In the present invention, "an aqueous medium" means a liquid containing water, such as a buffer and physiological saline. As examples of the buffer, tris(hydroxymethyl)aminomethane-HCl buffer (hereinafter referred to as "Tris-HCl buffer"), phosphate buffer, acetate buffer, succinate buffer, oxalate buffer, phthalate buffer, borate buffer, glycine buffer, barbital buffer, Good's buffer and the like may be enumerated.
As the sample containing sodium ions, any sample may be used as long as it is miscible with an aqueous medium. Biosamples such as whole blood and cells that are difficult to measure by the atomic absorption spectrometry, the flame photometry or the like can be measured by the present invention.
As the cation which competes with the sodium ion, an alkali metal ion, such as lithium, potassium, rubidium or cesium ion, or ammonium ion may be enumerated. These ions may be used independently or in combination. The suitable concentration of the cation which competes with the sodium ion is 130 mM - 0.5M for lithium ion, 20 mM-200 mM for potassium ion, 120 mM-500 mM for cesium ion, 20 mM-500 mM for rubidium ion and 50 mM-500 mM for ammonium ion.
As a source for lithium or potassium ions, chlorides, nitrates, sulfates, borohydride of these ions and the like may be enumerated. As a source for rubidium or cesium ions, chlorides of these ions and the like may be enumerated. As a source for ammonium ions, ammonium sulfate, ammonium chloride and the like may be enumerated.
The .beta.-galactosidase in the present invention may be any enzyme as long as it belongs to the enzyme number EC. 3.2.1.23. A .beta.-galactosidase derived from an animal, microorganism or plant, as well as an enzyme which is obtained by modifying such a .beta.-galactosidase with genetic engineering techniques are included.
As a substrate for .beta.-galactosidase, either synthetic or natural substrates may be used. For example, .beta.-D-galactoside, aryl .beta.-D-galactoside, alkyl .beta.-D-galactoside, 3,6-dihydroxyfluoran .beta.-D-galactoside, nitrophenyl .beta.-D-pyranoglycoside, nitrophenyl .beta.-D-galactoside, lactinol, lactose, 4-methylumbelliferyl .beta.-D-galactoside and the like may be enumerated. As an
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Miike Akira
Tadano Toshio
Umemoto Jun
Kyowa Medex Co., Ltd.
Leary Louise
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