Probe Compound for Detecting and Isolating Enzymes and Means and Methods Using the Same

• US 20120231972A1
• Filed: 03/19/2010
• Published: 09/13/2012
• Est. Priority Date: 03/19/2009
• Status: Abandoned Application

• Current Assignee: Helmholtz-Zentrum FüR Umweltforschung GmbH - UFZ (Bundesrepublik Deutschland), Institute of Catalysis
• Original Assignee: Institute of Catalysis
• Inventors: Chernikova, Tatyana, Ferrer, Manuel, Golyshin, Peter N., Golyshina, Olga V., Timmis, Kenneth N., Waliczek, Agnes, Beloqul, Ana, Guazzaroni, Maria E., Vieltes, Jose M., Pazos, Florencio, De Lacey, Antonio Lopez, Fernandez, Victor M.
• Application Number:
  US13/257,383
• Publication Number:
  US 20120231972A1
• Time in Patent Office: Days
• US Class Current: 506/11
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

The present invention relates to a probe compound that can comprise any substrate or metabolite of an enzymatic reaction in addition to an indicator component, such as, for example, a fluorescence dye, or the like. Moreover, the present invention relates to means for detecting enzymes in form of an array, which comprises any number of probe compounds of the invention which each comprise a different metabolite of interconnected metabolites representing the central pathways in all forms of life. Moreover, the present invention relates to a method for detecting enzymes involving the application of cell extracts or the like to the array of the invention which leads to reproducible enzymatic reactions with the substrates. These specific enzymatic reactions trigger the indicator (e.g. a fluorescence signal) and bind the enzymes to the respective cognate substrates. Moreover, the invention relates to means for isolating enzymes in form of nanoparticles coated with the probe compound of the invention. The immobilisation of the cognate substrates or metabolites on the surface of nanoparticles by means of the probe compounds allows capturing and isolating the respective enzyme, e.g. for subsequent sequencing.

Claims

1. A probe compound for detecting specific enzyme-substrate interactions comprising a transition metal complex and a reactive component of general formula (X):

His-L_His-TC-TC-L_TC-IC-IC-L_IC-His-His 

 

formula (X)

wherein His represents a histidine residue, TC represents a test component, IC represents an indicator component, and each of L_His-TC, L_TC-IC and L_IC-His independently represents optional linker components, wherein the reactive component is linked to the transition metal complex by the two histidine residues.

2. The probe compound according to claim 1, wherein the transition metal complex comprises a cobalt or copper atom.

3. The probe compound according to claim 1, wherein the transition metal complex further comprises a multidentate ligand.

4. The probe compound according to claim 1, wherein the transition metal complex comprises a nitrotriacetic acid cobalt(II) moiety.

5. The probe compound according to claim 3, wherein the multidentate ligand of the transition metal complex further comprises an anchoring component.

6. The probe compound according to claim 1, wherein the transition metal complex is a cobalt(II) complex of N_α

,N_α

-bis-(carboxymethyl)-L-lysine.

7. The probe compound according to claim 1, wherein the indicator component comprises a dye.

8. The probe compound according to claim 1, wherein the test component comprises a substrate selected from the group listed in Table 1.

9. The probe compound according to claim 1, wherein the indicator component comprises a fluorescence dye and wherein the linker component L_TC-IC comprises a quaternary ammine function.

10. A method of preparing a probe compound according to claim 1, which comprises the steps of:

(a) preparing a transition metal complex by reacting a salt of a transition metal with a ligand molecule;

(b) preparing a reactive component by

(i) linking a test component to a first histidine residue, optionally using a first linker component,

(ii) linking a dye component to a second histidine component, optionally using a second linker component, and

(iii) linking the test component to the dye component, optionally using a third linker component; and

(c) linking the reactive component to the transition metal complex using the first and second histidine residues.

11. An array for detecting enzymes comprising a plurality of different probe compounds according to claim 1.

12. A method for producing an array according to claim 11, comprising

(a) linking an anchoring component to the ligand of the transition metal complex of the probe compound, and

(b) arranging the different probe compounds in an array.

13. An isolation means comprising a nanoparticle and a probe compound according to claim 1.

14. A method for producing an isolation means according to claim 13, comprising

(a) linking an anchoring component to the ligand of the transition metal complex of the probe compound, and

(b) attaching the probe compound to a nanoparticle.

17. A method for detecting enzymes comprising contacting an analyte solution containing enzymes with the probe compound of claim 1 so that enzymes of the analyte solution are detected.

18. A method for detecting enzymes comprising contacting an analyte solution containing enzymes with the array of claim 11 so that enzymes of the analyte solution are detected.

19. A method for isolating enzymes comprising contacting a sample with the isolation means of claim 13 thereby isolating enzymes of the sample.

15-16. -16. (canceled)