Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1998-06-05
1999-05-18
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 4, 435 19, 4352831, 4352881, 422 55, 422 50, 422 8205, C12Q1/34;1/00
Patent
active
059050306
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to an enzyme reaction measuring method and its measuring apparatus in which a substrate solution and an enzyme solution are supplied into a reaction vessel so as to effect a reaction, and this enzyme reaction is continuously observed by infrared spectrometry.
BACKGROUND ART
Enzymes, which are a kind of catalyst produced by organisms, are mainly composed of proteins. Reaction materials of reactions catalyzed by the enzymes are known as substrates. For substantially all the reactions within living organisms, there exist enzymes corresponding thereto; and their enzyme reactions are performed under moderate conditions within the living organisms, thereby contributing to the maintenance of life. Accordingly, since a reduction in the action of an enzyme (enzyme activity) may cause a disease, it is prevalent in clinical tests to diagnose diseases by measuring activities of some kinds of enzymes in blood and urine.
Also, enzyme reactions are highly substrate-specific and are widely used as an energy-saving industrial process. For example, in fat and oil chemical industry, lipase is used for decomposing fats and oils. Lipase is an enzyme which hydrolyzes glycerol esters (triglycerides) of long-chain fatty acids. Lipase within alimentary canals mainly derives from the pancreas, though part thereof is secreted by the stomach and intestines. Lipase activity in blood has been known to rise in pancreatic diseases. Accordingly, the lipase activity in blood is measured to evaluate pancreatic diseases.
Measuring an enzyme reaction includes measuring quantitative and temporal changes in the substrate, which is a reaction material of the reaction catalyzed by the enzyme, and in a product generated by the reaction. Namely, the greater the ratio by which the substrate changes into the product, the higher the enzyme activity becomes. On the other hand, the smaller the ratio is, the lower the enzyme activity becomes. Also, when the change from the substrate to the product is measured over time, the reaction speed of the enzyme reaction can be analyzed.
Conventionally, in methods of measuring quantitative changes in the substrate and product, differences in physical or chemical properties of the substrate and product are utilized, so as to measure their respective quantitative changes. Examples thereof include: (1) a method in which the substrate or product is specifically caused to develop a color after the termination of the enzyme reaction, and is subjected to colorimetric determination, whereby the amount of increase or decrease in the substrate or product is determined (colorimetric determination method); (2) a method in which a compound labeled with a radioisotope (radioactive compound) is used as a substrate, the fact that the product generated by the enzyme reaction becomes a different radioactive compound is used to separate the radioactive compounds from each other, and the quantity of radioactivity is measured, whereby the amount of increase or decrease in the substrate or product is determined; (3) a fatty acid generated by the enzyme reaction is neutralization-titrated after the termination of the enzyme reaction (titration method); (4) a method in which the substrate is suspended in water, and the change in turbidity of the substrate solution upon decomposition of the substrate by the enzyme reaction is measured, whereby the amount of increase or decrease in the substrate or product is determined (turbidity measurement method); (5) a method in which, by utilizing spectroscopic differences between the substrate and product, wavelengths of light specifically absorbed by their respective materials (absorption wavelengths) are used, whereby the amount of increase or decrease in the substrate or product is determined from change in absorbance (spectroscopic measurement method); and the like. Among them, in particular, the spectroscopic measurement method and turbidity measurement method can continuously measure the enzyme reaction.
The above-mentioned measuring methods re
REFERENCES:
patent: 4022667 (1977-05-01), Myrick et al.
Imahori et al., "Dictionary of Biochemistry", K.K. Tokyo Kagaku Dojin (Tokyo), Apr. 10, 1984, refer to Lipase section.
"Furie Henkan Sekigaibunkoho (Fourier Transform Infrared Spectroscopy)," Gakkai Shuppan Center, (1985), pp. 163-171). (Partial Translation).
Matsumoto Hiroyuki
Okazaki Shigetoshi
Laboratory of Molecular Biophotonics
Leary Louise N.
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