Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Patent
1995-11-30
1998-06-23
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
435 4, 435 8, 435 15, 435 16, 435 18, 435 19, 435 21, 435 25, 435 26, 435 28, 436501, C12Q 166
Patent
active
057703914
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 filing of PCT/GB94/01163, filed May 27, 1994.
FIELD OF THE INVENTION
This invention relates to reagents for use in bioluminescence, and to their use.
BACKGROUND OF THE INVENTION
The background level of bioluminescence signal is often a problem when using the firefly luciferase reaction to measure adenosine 5'-triphosphate (ATP) levels deriving from prokaryotic cells, eukaryotic cells or both. Particular problems occur when the sample contains growth medium from a natural source, which is often the case in microbiological assays. Since such media are produced from living material, they tend to contain a significant amount of ATP. Most of the background bioluminescence signal arising from them can be removed by reaction with an ATPase enzyme such as potato apyrase or an ATP-utilising enzyme such as a kinase together with its co-substrate. However, a proportion of the signal always remains and can interfere with sensitive bioluminescence measurements. The source has always been thought of as non-microbial or non-cellular ATP which is somehow unavailable to ATPase enzymes, perhaps by reason of being sequestered.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that the residual background bioluminescence signal can be removed by reaction with certain other enzymes such as phosphodiesterase I from snake venom (E.C. 3.1.4.1; Enzyme Nomenclature 1984, Academic Press), acid phosphatase (E.C. 3.1.3.2) and alkaline phosphatase (E.C. 3.1.3.1). Likely candidates for this "non-degradable ATP" are substances structurally related to ATP that are not attacked by apyrase, or by other enzymes which have ATP as a substrate, yet give rise to light when acted upon by firefly luciferase. They may be true substrates of luciferase, or be acted upon by luciferase to form a true substrate such as ATP, or may break down to form ATP or another luciferase substrate by other unidentified means.
Flavine adenine dinucleotide (FAD) has been identified as one such substance. It occurs in many microbiological growth media and biological samples, is structurally related to ATP, appears to act as a light-emitting substrate for firefly luciferase, but is not degraded by apyrase; it is destroyed by snake venom phosphodiesterase I. Other such substances are adenosine 5'-tetraphosphate (p.sub.4 A) and other adenosine polyphosphates, diadenosine 5',5"'-P.sup.1,P.sup.4 -tetraphosphate (Ap.sub.4 A) and other diadenosine polyphosphates, and other dinucleoside polyphosphates such as Gp.sub.4 A and Gp.sub.5 A. It is likely that other, as yet unidentified, derivatives of ATP will have similar properties. A few of these have previously been suggested to act as low-efficiency light-emitting luciferase substrates; see Momsen (1978) Biochm. Biophys. Res. Commun. 84:816-822, and Sillero et al (1991) Eur. J. Biochem. 202:507-513.
These compounds mentioned may be destroyed by suitable enzymatic activity, e.g. snake venom phosphodiesterase I or phosphatases. Other enzymes exist which will act upon these nucleotide derivatives with various degrees of specificity. For example, adenosine tetraphosphatase (E.C. 3.6.1.14) can be used in the method of the invention: it hydrolyses the luciferase substrate adenosine 5'-tetraphosphate to give ATP, which will subsequently be destroyed by apyrase. Diadenosine 5',5"'-P.sup.1,P.sup.4 -tetraphosphatase (E.C. 3.6.1.17), which has been isolated from yellow lupin seeds (Jakubowski et al (1983) J. Biol. Chem. 258 (16):9982-9989), also produces ATP from its substrates and could be used in combination with apyrase in cases where part of the residual bioluminescence signal arises from Ap.sub.4 A.
A reagent to treat liquids, in order to reduce their response in a bioluminescent ATP assay, therefore comprises a combination of an ATP-degrading enzyme, such as potato apyrase, and one or more enzymes that degrade non-ATP luciferase substrates, together with additional divalent metal ions, such as Mg.sup.2+, if required. This combination of enzymes, or of enzyme activities in at
REFERENCES:
Biochemistry (by A. L. Lehninger, Publ. by Worth Publishers, Inc., New York, N.Y., 1970) p. 390.
Principles of Biochemistry (by White et al., Publ. by McGraw-Hill Co., New York, N.Y., 1973) pp. 729-730 and 482.
Traverso-Cori et al. (1970) Archives of Biochemistry and Biophysics, vol. 137, pp. 133-142.
Robinson et al. (1986) Archives of Biochemistry and Biophysics, vol. 248, No. 2, pp. 502-515.
Jakubowski, H., A. Guranowski (1983) "Enzymes Hydrolyzing ApppA and/or AppppA in Higher Plants" The Journal of Biological Chemistry 258(16):9982-9989.
Sillero, M.A.G. et al. (1991) "Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase" Eur. J. Biochem. 202:507-513.
Foote Nicholas Peter Martin
Grant Peter Leonard
Celsis International plc
Marschel Ardin H.
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