Method for determining the substrate specificity of an enzymatic activity and a device therefor

Abstract

The invention relates to a method for determining the substrate specificity of an enzymatic activity comprising the following steps: providing an assembly comprising a plurality of amino acid sequences on a planar surface of a support material, whereby the amino acids are directionally immobilised; contacting and/or incubating of an enzymatic activity with the assembly; and detection of a reaction between one of the amino acid sequences that are immobilised on the assembly and the enzymatic activity. According to the invention, during the reaction of the enzymatic activity with the assembly, a change in the molecular weight of at least one of the amino acid sequences takes place.

1 . A method for determining the substrate specificity of an enzymatic activity comprising the following steps: 
 Providing an assembly comprising a plurality of amino acid sequences on a planar surface of a support material wherein the amino acid sequences are directionally immobilised,    Contacting and/or incubating of an enzymatic activity with the assembly, and    Detecting a reaction between one or a plurality of amino acid sequences immobilised on the assembly and the enzymatic activity,    characterised in that during the reaction of the enzymatic activity with the assembly, a change in the molecular weight of at least one of the amino acid sequences takes place.    
     
     
         2 . The method according to  claim 1 , characterised in that the reaction is detected on or using the amino acid sequence immobilised on the surface of the support material.  
     
     
         3 . The method according to  claim 1  or  claim 2 , characterised in that the change in the molecular weight takes place by formation or cleaving of a covalent bond on one of the amino acid sequences, preferably on that amino acid sequence which reacts with the enzymatic activity.  
     
     
         4 . The method according to any one of  claims 1  to  3 , characterised in that the reaction is detected by detecting the change in the molecular weight.  
     
     
         5 . The method according to any one of  claims 1  to  4 , characterised in that the reaction is detected by a detection method selected from the group comprising autoradiography, plasmon resonance spectroscopy and fluorescence spectroscopy.  
     
     
         6 . The method according to any one of  claims 1  to  5 , characterised in that at least one of the amino acid sequences is a substrate for an enzymatic activity.  
     
     
         7 . The method according to any one of  claims 1  to  6 , characterised in that the assembly of amino acid sequences has at least one substrate for each of at least two different enzymatic activities.  
     
     
         8 . The method according to any one of  claims 1  to  7 , characterised in that the enzymatic activity is selected from the group comprising oxidoreductases, transferases, hydrolases, lyases and ligases, and especially is selected from the group comprising kinases, sulphotransferases, glycosyl transferases, acetyl transferases, farnesyl transferases, palmytyl transferases, phosphatases, sulphatases, esterases, lipases, acetylases and proteases.  
     
     
         9 . The method according to any one of  claims 1  to  8 , characterised in that the detection of a reaction between the amino acid sequences immobilised on the assembly and the enzymatic activity is repeated many times, preferably at intervals of time.  
     
     
         10 . The method according to any one of  claims 1  to  9 , characterised in that the enzymatic activity is determined in a sample and the sample is preferably selected from the group comprising urine, liquor, sputum, stool, lymph fluid, other body fluids, cell lysates, tissue lysates, organ lysates, extracts, raw extracts, purified preparations and unpurified preparations.  
     
     
         11 . The method according to any one of  claims 1  to  10 , characterised in that the surface is a non-porous surface.  
     
     
         12 . The method according to any one of  claims 1  to  11 , characterised in that the support material is glass.  
     
     
         13 . The method according to any one of  claims 1  to  12 , characterised in that the amino acid sequence is immobilised via a sulphur-comprising group on the surface.  
     
     
         14 . An assembly of a plurality of amino acid sequences on a surface wherein the amino acid sequences are directionally immobilised on the planar surface of a support material, characterised in that at least one of the amino acid sequences is a substrate for an enzymatic activity, wherein a change in the molecular weight takes place on the substrate as a result of the enzymatic activity.  
     
     
         15 . The assembly according to  claim 14 , characterised in that the change in the molecular weight takes place as a result of the formation or cleaving of a covalent bond on the substrate.  
     
     
         16 . The assembly according to  claim 14  or  claim 15 , characterised in that the assembly of amino acid sequences has at least one substrate for each of at least two different enzymatic activities.  
     
     
         17 . The assembly according to any one of  claims 14  to  16 , characterised in that the planar surface is a non-porous surface.  
     
     
         18 . The assembly according to  claims 14  to  17 , characterised in that the support material is selected from the group comprising silicates, ceramic, glass, metals and organic support materials.  
     
     
         19 . The assembly according to any one of  claims 14  to  18 , characterised in that the amino acid sequences are selected from the group comprising peptides, oligopeptides, polypeptides and proteins as well as their respective derivatives.  
     
     
         20 . The assembly according to any one of  claims 14  to  19 , characterised in that each amino acid sequence or group of amino acid sequences has a defined arrangement relative to another amino acid sequence or groups of amino acid sequences.  
     
     
         21 . A support comprising an assembly according to any one of the preceding claims.  
     
     
         22 . The support according to  claim 21 , characterised in that the support comprises a base support material.  
     
     
         23 . The support according to  claim 21  or  22 , characterised in that the assembly of a plurality of amino acid sequences is arranged on one or a plurality of surfaces of the support.  
     
     
         24 . The support assembly comprising at least two supports according to any one of  claims 21  to  23 , wherein respectively two supports are separated by a gap.  
     
     
         25 . The support assembly according to  claim 24 , characterised in that at least one assembly on a first support is facing at least one assembly on a second support.  
     
     
         26 . The support assembly according to  claim 24  or  claim 25 , characterised in that the gap has a width of around 0.01 mm to 10 mm, preferably around 0.1 mm to 2 mm, and more preferably around 0.5 mm to 1 mm.  
     
     
         27 . The use of an assembly according to any one of  claims 14  to  20  and/or a support according to any one of claims  21  to 23 and/or a support assembly according to any one of  claims 24  to  26  in a method according to any one of  claims 1  to  13 .