Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1988-08-18
1991-07-30
Raymond, Richard L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 24, 536 24, 548178, 530330, 530331, C07D27768, C07D41704
Patent
active
050359991
DESCRIPTION:
BRIEF SUMMARY
The invention relates to aminoluciferin derivatives, processes for their production and to the use thereof for the determination of enzyme activities.
In the common analytical methods for determining enzyme activities, for instance in a biological material, fluorogenic and chromogenic substrates are used from which a leaving group can be cleaved off with the help of the enzymes.
Such a leaving group can be for instance a dye. The dyes which are developed in the course of said indicator reaction can be measured photometrically. Therefore it is possible to determine their concentration and to deduct from the data obtained informations with respect to the enzymatic activity, see "Methods of Enzymatic Analysis, Verlag Chemie, Vol. 1-11, Weinheim, BergstraBe (1984), editor H. U. Bergmeyer.
As an example for such a leaving group the concentration of which can be photometrically determined the following group is mentioned: ##STR2##
In case one uses the above mentioned fluorogenic substrate one can then photometrically determine the leaving group. As an example for the leaving group of a fluorogenic substrate the following group can be mentioned: ##STR3## With the known substrates it is possible to determine or measure, respectively, enzyme concentrations down to about 5 ng per test. In this case it is referred to the enzymatic activity (U). From the specific activity (U/mg) the amount of enzyme can be deducted.
The enzymatic activity (U) is defined as .mu.mol/min; mainly measured at 25.degree. C. Said enzymatic activity is also named catalytic activity and is expressed according to SI-units as Katal (kat=mol/s). The catalytic concentration (catalytic activity per volume) is expressed as kat/l.
By the present invention there are provided new substrates. With the help of said substrates it is possible to surprisingly increase the sensitivity of the analytical methods mentioned above in order to determine enzyme activities. It is for instance possible to determine enzyme concentrations down to about 10 to 100 fg per test. This means that the detection limit has been lowered. The sensitivity depends of course on the enzyme investigated and the substrate used.
It is common to all substrates as provided according to the present invention that the enzymes tested are capable of cleaving off or liberating, respectively, D-aminoluciferin of the following formula V ##STR4## from said substrates.
Said aminoluciferin (V) therefore represents a leaving group as it has been explained in the beginning.
The liberated aminoluciferin can be luminometrically detected even in smallest concentrations. For this purpose said aminoluciferin is reacted with the enzyme luciferase of the fire-fly Photinus pyralis or of the fire-fly Photinus plathiophthalamus or of the luciferase of other species or chemically or genetically modified luciferases in the presence of ATP+MgCl.sub.2. In the course of said reactions photones are emitted; i.e. in the course of the reaction with the enzyme of the fire-fly Photinus pyralis at 605 nm and in the course of the reaction with the enzyme of the fire-fly Photinus plathiophthalamus at 549 or 570 nm, or wavelength corresponding to the used liciferin/luciferase system, respectively. The emission at 549 nm takes place if the enzyme originates from the dorsal organ of the fire-fly mentioned whereas the emission at 570 nm takes place if the enzyme originates from the ventral organ.
The above described is a bioluminescence. The light emitted is measured luminometrically.
For further details it is referred to the following literature: "Luminometry" by K. Wulff in "Methods of Enzymatic Analysis", Vol. I (editor: H. U. Bergmeyer), pages 340-368, Verlag Chemie, Weinheim, BergstraBe (1983) and Journal of the American Chemical Society 88, 2015-2018, 1966 by E. H. White et al.
By the data obtained the enzyme concentration of the investigated enzyme can be determined. The exact execution of said determination with the help of a "bioluminescence cocktail" as well as the calibration are explained further in the examples.
T
REFERENCES:
White et al, JACS, vol. 88, No. 9 (5-1960), pp. 2015-2019.
Geiger Reinhard
Miska Werner
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