Methods and compositions of enzymatic cycling based assays for myeloperoxidase

Abstract

The present invention provides a method for assaying myeloperoxidase activity. In the assay, a sample containing myeloperoxidase or suspected of containing myeloperoxidase is contacted with substrate including serine, hydrogen peroxide, and a halide. If myeloperoxidase is present in the sample, serine is converted into glycolaldehyde, which is further converted into glycolate by a glycoaldehyde converting enzyme. The method then utilizes a cycling reaction system between glycolate and glyoxylate to generate a detectable signal that corresponds to the myeloperoxidase activity. Kits for assaying myeloperoxidases based on the same principle are also provided.

1 . A kit for assaying myeloperoxidase, said kit comprises: serine, hydrogen peroxide, a halide, a glycolaldehyde converting enzyme which catalyzes conversion of glycolaldehyde to glycolate, a first electron acceptor in its oxidized form, a glycolate converting enzyme which catalyzes conversion of glycolate to glyoxylate, a second electron acceptor in its oxidized form, and a glyoxylate converting enzyme which catalyzes conversion of glyoxylate to glycolate, and a third electron acceptor in its reduced form, wherein the second electron acceptor and the third electron acceptor are different.  
   
   
       2 . The kit of  claim 1 , wherein the glycolaldehyde converting enzyme is a glycolaldehyde dehydrogenase and the first electron acceptor is selected from the group consisting of NAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl-NADP + .  
   
   
       3 . The kit of  claim 1 , wherein the glycolate converting enzyme is a glycolate reductase and the oxidized form of the second electron acceptor is selected from the group consisting of NAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl-NADP + , wherein the glyoxylate converting enzyme is a glyoxylate reductase and the reduced form of the third electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH(R)-lactate, 2,6-dichloroindophenol, and phenazine methosulfate.  
   
   
       4 . The kit of  claim 1 , wherein the glycolate converting enzyme is a glycolate oxidase and the oxidized form of the second electron acceptor is O 2 , and the glyoxylate converting enzyme is a D-glycerate dehydrogenase and the reduced form of the second electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH.  
   
   
       5 . The kit of  claim 1 , wherein both the glycolate converting enzyme and the glyoxylate converting enzyme are a glycolate reductase or a glyoxylate reductase, and wherein the oxidized form of the second electron acceptor is selected from the group consisting of NAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl-NADP + , and the reduced form of the third electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH.  
   
   
       6 . The kit of  claim 1 , wherein the glycolate converting enzyme is glycolate reductase.  
   
   
       7 . The kit of  claim 1 , wherein the glycolate converting enzyme is glycolate oxidase.  
   
   
       8 . The kit of  claim 1 , wherein the glycolate converting enzyme is glyoxylate reductase.  
   
   
       9 . The kit of  claim 1 , wherein the glyoxylate converting enzyme is glycerate dehydrogenase.  
   
   
       10 . The kit of  claim 1 , wherein the glyoxylate converting enzyme is glyoxylate reductase.  
   
   
       11 . The kit of  claim 1 , wherein the glyoxylate converting enzyme is glycolate reductase.  
   
   
       12 . The kit of  claim 2 , wherein the glycolate converting enzyme is glycolate reductase and the glyoxylate converting enzyme is glyoxylate reductase.  
   
   
       13 . The kit of  claim 2 , wherein the glycolate converting enzyme is glycolate oxidase and the glyoxylate converting enzyme is D-glycerate dehydrogenase.  
   
   
       14 . The kit of  claim 2 , wherein the glycolate converting enzyme and the glyoxylate converting enzyme are both glycolate reductase.  
   
   
       15 . The kit of  claim 1 , wherein the halide is chloride.  
   
   
       16 . The kit of  claim 2 , wherein the halide is chloride.  
   
   
       17 . The kit of  claim 1 , said kit further comprises an instruction indicating use for prognosis and/or diagnosis of a disease.  
   
   
       18 . The kit of  claim 15 , wherein the disease is selected from the group consisting of atherosclerosis, stroke, multiple sclerosis, Alzheimer's disease, lung cancer, leukemia, and infection.