DE19602662C2 - Method for the bioluminometric determination of pyrophosphate - Google Patents

Description

The invention relates to a method for the use of L-luciferin in bioluminometric Measurements.

The emission of light in the course of chemical catalysis is called bioluminescence designated. In the presence of adenosine triphosphate (ATP), luciferase (Firefly luciferase EC.1.13.12.7) the oxidative decarboxylation of D-luciferin to oxyluciferin. This reaction is associated with the emission of light. Because light is very sensitive can be measured, this method is generally used for the determination of small amounts of ATP (Gould, S. and Subramani, S. (1988) Analytical Biochemistry, 175, 5-13). the The main application of the bioluminometric ATP measurements is the detection of bacterial Contaminants (food chemistry, military) as well as the analysis of biochemical reactions (biological and medical research) (Kricka, L. (1988) Analytical Biochemistry, 175, 14-21).

The catalysis of the luciferase is stereospecific and a light emission can therefore only in Presence of D-luciferin, but not when using L-luciferin, can be measured (Seliger, H., McElroy, W., White, E. and Field, G. (1961) Proceedings of the National Academy of Science, 47, 1129-1134; McElroy, W. and Seliger, H. (1962) Federal Proceedings, 21, 1006-1012). Both isomers can be synthesized (Bowie, L. (1978), Methods in Enzymology, Vol. LVII, 15-28). Due to the structural similarity, L- Luciferin as a competitive inhibitor of D-luciferin. This fact is used by commercial "ATP momtoring kits" used to stabilize the luciferase activity and provides at the same time represents the most important practical application of L-luciferin (Lundin, A. (1982) Luminescent Assays: Perspectives in Endocrinology and Clinical Chemistry, Eds. Serio, M. and Pazzagli, M., Raven Press, New York).

The invention is based on the problem of the stereospecificity of the bioluminometric Bypassing the reaction and thus increasing the scope of bioluminometric measurements expand.

According to the invention, this object is achieved by the following process steps:

• - Luciferase is preincubated with adenosine triphosphate, L-luciferin and pyrophosphatase,
• - the reaction is triggered by adding pyrophosphate and a flash of light is emitted, which enables a qualitative and quantitative pyrophosphate measurement.

It has surprisingly been found that after the addition of pyrophosphate to the preincubated other components emit a strong flash of light, which is a qualitative and allows quantitative luminometric measurement.

The scope of the invention also includes the use of the method according to the invention for bioluminometric determination of pyrophosphate.

The advantage of the invention lies in the expansion of the bioluminometric measurement spectrum Measurements. With the same high sensitivity, it is possible to add a new class of substances, Pyrophosphates, measure. Analogous to the bioluminometric adenosine triphosphate measurement This creates the possibility of coupling chemical reactions to increase production or record the consumption of pyrophosphate. Pyrophosphate is formed e.g. B. in the course of Activation of cells by hormones, besides it plays an important role in the process of hormonal control of mitochondrial activity.

The invention is described below using an exemplary application example.

Example:

With regard to pH, temperature and buffer composition, the preincubation takes place Luciferase with adenosine triphosphate (ATP) and L-luciferin under standard conditions (such as z. B. in Lembert, N. and Idahl, L.-Å., (1995) Biochemical J., 305, 929-933). That continuous detection of light emission is possible with commercially available luminometers possible.

Fig. 1 shows by way of light-emission-time curve the effect of pyrophosphate addition to the light emission from luciferase.

The open circles denote the course of the reaction, measured as light emission, from Luciferase (0.1 µM) in the presence of adenosine triphosphate (ATP) (100 µM) and L- Luciferin (1 µM). At the time of 6 min, pyrophosphate (PPi) (50 µM) is added to this solution admitted. The absence of any significant light emission among these The reaction conditions correspond to the state of the art.

The solid line denotes the course of the reaction in the case of the invention Method. Here, luciferase (0.1 µM) is used in the presence of adenosine triphosphate (ATP) (100 µM) and L-luciferin (1 µM) preincubated with pyrophosphatase (0.2 U / ml). To the At 6 min, pyrophosphate (PPi) (50 μM) is added to this solution. The one with it The flash of light that occurs is clearly visible.

A flash induction was already measured within the following concentration intervals:

Luciferase: 5 nM to 0.1 µM Adenosine triphosphate: 10 to 1,000 µM L-luciferin: 0.1 to 2 µM Pyrophosphatase: 0.2 to 5 U / ml Pyrophosphate: 10 to 100 µM

Both the buffer composition and the concentrations of the reactants can be used in can be varied within wide limits.

Claims (1)

1. A method for the bioluminometric determination of pyrophosphate, characterized by the following process steps:

• 1. Luciferase is preincubated with adenosine triphosphate, L-luciferin and pyrophosphatase,
• 2. The reaction is triggered by adding pyrophosphate and a flash of light is emitted, which enables a qualitative and quantitative pyrophosphate measurement.