Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1990-01-05
1993-10-12
Griffin, Ronald W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 772, 435968, 436546, 436800, 424 71, C12Q 134
Patent
active
052524620
DESCRIPTION:
BRIEF SUMMARY
The technical field of the invention comprises the use of certain specific fluorescent or fluorogenic substrates for enzyme activity measurements.
A fluorescent organic substance usually carries a chromophore i.e. a structure strongly absorbing light of the UV or visible regions. The term "chromophore" therefore refers to a system of double and/or triple bonds which are conjugated with each other or are linked to bridges consisting of hetero atoms with free electrons (in the first place oxygen, nitrogen, sulfur) and hetero atoms bound directly to the system. Often the double bonds will form aromatic ring systems. Changes in the number of double and triple bonds, their relative positions with respect to each other, and the hetero atom binding conditions will strongly affect the light absorbent properties. The effects of groups bound aliphatically are much weaker, and such groups are therefore not regarded as being parts of chromophores.
The rate and course of an enzymatic reaction are determined by the enzyme and the substrate and by a number of other substances which are either required in order for enzyme activity to occur or are apt to strongly influence that activity. Examples of such substances are cofactors like coenzymes and cosubstrates, allosteric effectors and inhibitors. For cases where a sample is suspected to contain one or more of these substances techniques have been developed where the particular substance(s) suspected to be present is/are detected by addition of the remaining substances and measurement of enzyme activity. For clinical samples, various types of enzyme activity have been correlated with specific disorders. Within the field of immunochemical analyses, assays for an analyte with the aid of one or more immune reactants carrying analytically detectable groups have been carried out by using marker groups consisting of some of such substances affecting the course of the enzymatic reaction, including also the enzyme itself.
Fluorescent and fluorogenic substrates were employed already in early types of enzyme activity determination methods. Their advantages resided in the first place in the low detection limit which is characteristic of fluorescent substances. In one of these cases, the substrate and product had virtually identical fluorescent properties; in order to enable substrate conversion to be measured it was necessary to separate the product from the substrate. See for example J. Appl. Biochem. 5 (1983) 399-403 where insoluble casein with covalently bound europium chelate was used for determining the proteolytic activity by means of time resolved fluorescence spectrometry. In the other case a fluorescent or fluorogenic substrate was employed which had fluorescent properties differing from those of the product obtained. This difference could be a difference in excitation as well as a difference in emission characteristics. Basically it was not necessary to separate the substrate from the product. The substrates were constructed in a manner such that the chromophoric structure was formed, destroyed or changed as a consequence of the enzymatic reaction. Both of these types of substrate groups have been synthesized for enzymes belonging to the various different main groups of enzymes (oxidoreductases, transferases, ligases, hydrolases, lyases and osomerases). See e.g. Enzymes; Ed: Dixon, M. et al; Longham Group Ltd, London 1979.
In biological samples fluorescence measurements are thwarted by background fluorescence from e.g. proteins. Due to this background, sensitivity will be found to be low when specific substances are to be determined. It is known that this disturbing effect can be avoided if the fluorescent marker (for example substrate/product) is chosen such that its emission maximum and/or excitation maximum are clearly distinct from the background fluorescence. An alternative approach has been to choose a marker having a long fluorescent decay time as compared to that of the background fluorescence, and to then employ time resolved fluorescence spectrometry for measuring
REFERENCES:
patent: 4837169 (1989-06-01), Toner
patent: 4857475 (1989-08-01), Dakubu
patent: 5011910 (1991-04-01), Marshall et al.
Carlsson Jan
Drevin Hakan
Hemmila Ilkka
Kwiatkowski Marek
Lovgren Timo
Griffin Ronald W.
Pharmacia AB
Wallac Oy
Webber Pamela S.
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