Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Patent
1980-07-15
1982-10-12
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
435 14, 435 25, 435 28, 435817, 23230B, 204 1T, 204195B, C12Q 160, C12Q 154, C12Q 128
Patent
active
043539831
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The measurement of hydrogen peroxide concentration in aqueous systems is important in many cases such as, for instance, when using H.sub.2 O.sub.2 as an oxidant for the treatment of effluents (see W. H. KIBBEL, Peroxide Treatment of Industrial Waste Waters, Industrial Water Engineering, August-September 1976) and in medical diagnostic analysis. Thus, in connection with the medical aspect, many analytical techniques exist based on enzymatic oxidation reactions which involve the quantitative production of H.sub.2 O.sub.2. Such techniques are particularly valuable because of the highly selective and sensitive behavior of some enzymatic systems toward specific substrates.
Thus, it is well-known to use an "oxidase" for catalyzing the quantitative oxidation of a substrate with the formation of a corresponding amount of hydrogen peroxide. This H.sub.2 O.sub.2 can be thereafter measured by another enzymatic reaction in which an indicator dye is oxidized by this H.sub.2 O.sub.2 in the presence of a peroxidase whereby the intensity of the color developped is a measure of the amount of the H.sub.2 O.sub.2 present in the system and, consequently, a measure of the amount of the substrate originally present which generated the said H.sub.2 O.sub.2 upon oxidation.
The "oxidase" enzymes suitable for such determination are usually named from the type of substrate they can act upon; thus, glucose oxidase specifically catalyzes the oxygen oxidation of glucose into gluconic acid with liberation of H.sub.2 O.sub.2 ; cholesterol oxidase acts similarly toward cholesterol, etc.
DESCRIPTION OF THE PRIOR ART
Examples of oxidases can be found in the literature familiar to those skilled in the art, namely in "Enzyme Nomenclature Recommendations" (1972), International Union of Biochemistry, Elsevier Scientific Publishing Co.
There exists also several different peroxidases which are suitable for catalyzing such oxidations by H.sub.2 O.sub.2 among which the peroxidases of horse-radish and of Japanese-radish are well-known. Various peroxidases are described by BOYER et al. in "The Enzymes" Vol. 8 (1963), Academic Press. Moreover, hemoglobin, or rather some of its constituents, can also act as a peroxidase in some cases.
Besides the method mentioned heretofore, several other enzymatic routes can be used for determining substrates in biological fluids, for instance glucose in blood or urine. Thus, besides titrating the H.sub.2 O.sub.2 formed by the glucose oxidase catalyzed oxidation reaction by quantitatively oxidizing a dye in the presence of peroxidase, the amount of said H.sub.2 O.sub.2 can also be measured polarographically by means of an electrode equipped with a semi-permeable membrane. Otherwise, instead of measuring the H.sub.2 O.sub.2 formed, the oxygen consumed in the oxidation of glucose can also be measured with an electrode provided with a gas permeable membrane, e.g. an electrode such as the well-known "CLARK" electrode.
However, the above analytical procedures are not free from some drawbacks. For instance, the colorimetric method requires that the sample be inherently colorless and not turbid, otherwise significant measurement errors may result. Also, in connection with the electrometric methods involving membranes, the latter will require to be maintained carefully, otherwise bacterial contamination may occur with eventual spoiling of the electrodes. It is also desirable to have a general system and method enabling to determine either organic substrates that will generate H.sub.2 O.sub.2 upon enzymatic oxidation, or the H.sub.2 O.sub.2 formed during this oxidation, or even the enzymes themselves that act as catalysts in such reactions.
OBJECT OF THE INVENTION
The present invention proposes to remedy the above drawbacks and provide a versatile analytical method and apparatus.
SUMMARY OF THE INVENTION
The present invention utilizes the known phenomenon that some fluoro-compounds are oxidized by H.sub.2 O.sub.2 in the presence of peroxidase with the quantitive splitting of the fluorine a
REFERENCES:
patent: 3483112 (1969-12-01), Ross
patent: 3595755 (1971-07-01), Hurtel
patent: 3902970 (1975-09-01), Levin
patent: 4098574 (1978-07-01), Dappen
Hughes, et al., "Studies in Peroxidase Action, Part IX, Reactions Involving the Rupture of C--F, C--Br, and C--I Links in Aromatic Amines", J. Chem. Soc., (1954), pp. 4630-4634.
Saunders et al., Peroxidase, Buttersworths & Co., Washington, (1964), pp. 24, 25, 204, 205.
Battelle (Memorial Institute)
Ross Karl F.
Wiseman Thomas G.
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