Protease detection assay

a protease activity and assay technology, applied in the direction of electrolysis components, material analysis by electric/magnetic means, biochemistry apparatus, etc., can solve the problem of lack of sensitivity of methods, and achieve the effect of facilitating detection and efficient electron transfer

Inactive Publication Date: 2010-12-23
ATLAS GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides a method for detecting protease activity in a sample by using a protease substrate labelled with an electrochemically active marker. The method can quantify the relative proportions of degraded and non-degraded substrate. The invention also provides methods for labelling proteins, peptides, and amino acids via carboxyl groups, which allows for the production of labelled substrates with a free, underivatised amino terminal. This is useful for developing working assays for certain amino peptidases which are a class of proteases that degrade peptides from the amino terminal.

Problems solved by technology

Such methods often lack sensitivity, require sampling to obtain kinetic data and depend on quantitative precipitation for accurate results.

Method used

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Examples

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example 1

ltammetry

[0123]This Example describes the cyclic voltammetry method used in Examples 5 to 11 below.

[0124]The low volume cell of FIG. 1 was filled with approximately 10 ml sodium chloride solution (100 mM). A 200 μl aliquot of the sample for analysis was placed in the glass sample chamber which was then placed in the low volume cell along with the reference and counter electrodes. The electrodes were connected to the Autolab electrochemical workstation and differential pulse voltammetry carried out using the parameters described in Table 1. Prior to analysis the glassy carbon working electrode was polished (using BAS polishing kit catalogue number MF-2060) followed by conditioning. Electrode conditioning consists of cyclic voltammetry, sweeping between ±1 volt in the appropriate background buffer.

TABLE 1Parameters for differential pulse voltammetry:Parameter:Anodic sweepConditioning potential (V)0Conditioning duration (s)10Deposition potential (V)0Deposition duration (s)0Equilibratio...

example 2

Synthesis of N-Hydroxysuccinimide Ester of Ferrocenecarboxylic Acid

[0125]Ferrocenecarboxylic acid (303 mg, 1.32 mmol) and N-hydroxysuccinimide (170 mg, 1.47 mmol) were dissolved in dioxane (15 ml) and added with stirring to a solution of dicyclohexylcarbodiimide (305 mg, 1.48 mmol) in dioxane (3 ml). The mixture was stirred at room temperature for 24 hours during which time a precipitate was formed. The precipitate was removed by filtration, solvent was removed from the filtrate in vacuo and the resulting, solid purified by silica gel column chromatography, eluting with 8:2 petrol:ethyl acetate. Yield 320 mg, 74%.

example 3a

Synthesis of Ferrocene Carbonyl Azide

[0126]Ferrocene carbonyl azide was prepared from ferrocenecarboxylic acid by reaction with oxalyl chloride and sodium azide.

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Abstract

The invention provides methods and compounds for detecting protease activity in a sample solution comprising contacting the sample solution with a protease substrate labeled with an electrochemically active marker, providing conditions under which any protease which may be present in the sample may degrade the protease substrate and electrochemically determining information relating to the electrochemically active marker.

Description

FIELD OF THE INVENTION[0001]The invention relates to methods for the detection or assessment of protease activity, and to substrates and apparatus for use in such methods.BACKGROUND OF THE INVENTION[0002]Protease enzymes are involved in a variety of biological phenomena, for example, in protein activation and cell signalling. Protease activity plays a key role in processes such as blood clotting, apoptosis and hormone regulation. Proteases are also essential to the function of a variety of viral and microbial pathogens. There is an increasing interest in the development of protease inhibitors for use as therapeutic agents.[0003]The determination of protease activity in biological samples is important in the analysis of processes such as apoptosis, in the screening of potential protease inhibitors and in the monitoring of sample purity, for example during protein purification. Protease enzymes hydrolyse amides and esters to produce peptides, single amino acids or labelled amino acid ...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12Q1/00C10L1/30C07F15/00G01N27/26C12Q1/37G01N27/48G01N33/573
CPCC12Q1/37
InventorBRAVEN, HELENKEAY, RUSSELLFLOWER, STEPHEN
OwnerATLAS GENETICS