DESC:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of estimating enzyme activity, in specific, relates to a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis with zymography, which does not disturb a cysteine switch and makes detection easier.
Background of the invention:
[0002] Biological enzymes catalyze a wide variety of chemical reactions in all living organisms. They are essential for all life processes, including digestion, metabolism, energy production, and cell signaling. Often times, it is important to understand the activity of various biological enzymes in order to evaluate any malfunctions that could result in human disease or imbalances in homeostasis. Rapid extraction, purification, and evaluation of enzymes and their activity is still a big challenge today.

[0003] MMPs (matrix metalloproteinases), are a family of biological enzymes that play a role in breaking down the extracellular matrix (ECM). The ECM is a network of proteins and other molecules that provides support and structure to tissues. MMPs are involved in a variety of physiological and pathological processes, including wound healing, tissue remodeling, inflammation, and cancer. MMPs are activated by a cysteine switch, which controls the activity of protein. There are a number of ways to identify MMPs. One common method is zymography. Zymography is a gel electrophoresis technique that is used to detect enzymatic activity in a gel. Zymography can be used to detect any protease that can cleave the substrate embedded in the gel. To identify MMPs (matrix metalloproteinases) using zymography, the enzyme substrate is embedded in a polyacrylamide gel. The sample is then loaded onto the gel and electrophoresed. If MMPs are present in the sample, they will degrade the substrate in the gel, resulting in a clear band. The gel is then stained to visualize the bands.

[0004] Zymography is mostly used to detect the activities of the proteolytic enzyme. It is a method used for screening, identification, and characterization of proteinases. Rather than visualizing all proteins separated on the gel using a general staining method, zymography recognizes a particular group of enzymes which remain active after the electrophoresis. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis which separates proteins based on their molecular weight.

[0005] SDS-PAGE, or sodium dodecyl sulfate–polyacrylamide gel electrophoresis, is a technique used to separate proteins based on their molecular weight. It is a widely used technique in biochemistry, molecular biology, and forensics. SDS-PAGE works by first denaturing the proteins, which unfolds them and removes their secondary, tertiary, and quaternary structure. This is done by treating the proteins with sodium dodecyl sulfate (SDS), a detergent that binds to the proteins and coats them with a negative charge. The denatured proteins are then loaded into a polyacrylamide gel and subjected to an electric field.

[0006] Proteins migrate through the gel at different speeds depending on their molecular weight. Smaller proteins move faster through the gel than larger proteins, because they face less resistance. The proteins are separated into bands, with the smallest proteins at the bottom of the gel and the largest proteins at the top. After the proteins have been separated, the gel can be stained to visualize the bands. A common stain used for SDS-PAGE is Coomassie blue, which stains all proteins blue. Once the gel has been stained, the molecular weight of the proteins can be estimated by comparing the bands to the positions of known molecular weight standards. However in SDS technique the cysteine switch is disturbed and break the protein structure, which is a major disadvantage in detecting the active molecules. The SDS-PAGE gels are carried through a process of renaturation which may or may not yield a completely functional MMP. The MMP samples are completely destroyed while boiling and become inactive due to the dead enzyme (MMPs).

[0007] By addressing the above mentioned problems, there is a need for a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis process with zymography, which helps in rapid detection of enzyme activity of various proteinases without lengthy procedures of enzyme extraction, purification and evaluation. There is also a need for a method which does not disturb a cysteine switch and make detection of enzymes easier. There is also a need for a method that overcomes the challenges like extraction, purification and evaluation of enzymes. There is also a need for a method for detecting active molecules of the bacterial or plant or mammalian cell proteasomes without interrupting the zymogen in the protein structure. There is also a need for a method for estimating enzyme activity, which improves detection capabilities of malfunctioning enzymes in clinical settings and enhances the pace of biomedical research. There is also a need for a method, which is highly beneficial for testing the unpurified samples such as blood-plasma.
Objectives of the invention:
[0008] The primary objective of the present invention is to provide a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis process with zymography, which helps in rapid detection of enzyme activity of various proteinases without lengthy procedures of enzyme extraction, purification and evaluation.

[0009] Another objective of the present invention is to provide a method, which does not disturb a cysteine switch while activating MMPs (matrix metalloproteinases) and makes detection of enzymes easier.

[0010] Another objective of the present invention is to provide a method that overcomes the challenges like extraction, purification and evaluation of enzymes.

[0011] Yet another objective of the present invention is to provide a method for detecting active molecules of the bacterial or plant or mammalian cell proteasomes without interrupting the zymogen in the protein structure.

[0012] Another objective of the present invention is to provide a method for estimating enzyme activity, which improves detection capabilities of malfunctioning enzymes in clinical settings and enhances the pace of biomedical research.

[0013] Further objective of the present invention is to provide a method, which is highly beneficial for testing the unpurified samples such as blood-plasma.
Summary of the invention:
[0014] The present disclosure proposes a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis with zymography. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0015] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis process with zymography, which helps in rapid detection of enzyme activity of various proteinases without lengthy procedures of enzyme extraction, purification and evaluation.

[0016] According to one aspect, the invention provides a method for estimating enzyme activity by combining a native polyacrylamide gel electrophoresis process with zymography. At one step, one or more blood samples are collected from at least one patient without mixing with Ethylenediaminetetraacetic acid (EDTA) to avoid any interference with a zymogram.

[0017] At another step, a plasma is separated from the blood sample by centrifuging the blood samples at 5000 rpm for at least 20 min in order to obtain extracellular enzymes. At other step, the separated plasma is then transferred into conduit without disturbing a cell pellet upon centrifugation and the conduit filled with plasma is stored in an ice container.

[0018] At another step, a pair of gel samples containing 12 weight percentage of separating gel and 5 weight percentage of stacking gel are prepared using a gel electrophoresis unit and then the pair of gel samples are solidified to attain solidified gel samples. The separating gel and stacking gel contains a gelatin of about 1 mg/ml.

[0019] At other step, the solidified gel samples are loaded with equal volume of plasma mixed with a native gel loading dye solution upon solidification and then the solidified gel samples are loaded into wells for performing a native PAGE (polyacrylamide gel electrophoresis). The native PAGE (polyacrylamide gel electrophoresis) is performed in the 1X Tris –Glycine buffer at 120 volts for a time period of at least 1 hour.

[0020] At another step, the solidified gel samples are removed from the wells without any tearing and the solidified gel is incubated in a buffer solution for a time period of 24 hours at a temperature of about 37 degrees. The buffer solution used is about a volume 50 ml, which contains 2.5 ml of 50 mM Tris Hcl, 125 µl of 5 mM Cacl2, 0.1 M and 0.5 µl of Zncl2.

[0021] At other step, the incubated gel from the buffer solution is removed and rinsed gently with deionized water for removing any remnants of the buffer solution. Further at other step, the incubated gel is stained with at least one day overnight at a room temperature of 37 degrees for obtaining a stained gel and the stained gel is rinsed with deionized water to remove at least one dye until clearance zones around one or more protein bands are observed clearly. The at least one dye includes a coomassie brilliant blue dye.

[0022] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0023] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0024] FIG. 1 illustrates a flowchart of a method for estimating enzyme activity by combining a native polyacrylamide gel electrophoresis process with zymography, in accordance to an exemplary embodiment of the invention.

[0025] FIG. 2A illustrates a panel representing a control Native Zymo performed using a purified enzyme, in accordance to an exemplary embodiment of the invention.

[0026] FIG. 2B illustrates a panel representing a control Native Zymo performed using healthy volunteer plasma and patient plasma, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0027] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0028] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis process with zymography, which helps in rapid detection of enzyme activity of various proteinases without lengthy procedures of enzyme extraction, purification and evaluation.

[0029] According to one exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for estimating enzyme activity by combining a native polyacrylamide gel electrophoresis process with zymography.

[0030] Zymography is a gel electrophoresis technique that is used to detect enzymatic activity in a gel. Zymography can be used to detect any protease that can cleave the substrate embedded in the gel. To identify matrix metalloproteinases (MMPs) using zymography, the enzyme substrate is embedded in a polyacrylamide gel. The sample is then loaded onto the gel and electrophoresed. If MMPs are present in the sample, they will degrade the substrate in the gel, resulting in a clear band. The gel is then stained to visualize the bands.

[0031] Native PAGE, or native polyacrylamide gel electrophoresis, is a technique used to separate proteins based on their native conformation, which includes their size, charge, and shape. It is a type of gel electrophoresis, which is a technique that uses an electric field to separate molecules based on their size and charge.

[0032] Native PAGE is different from SDS-PAGE in that the proteins are not denatured before they are loaded onto the gel. This means that the proteins retain their native structure and interactions. Native PAGE is often used to study multimeric proteins, which are proteins that are made up of two or more subunits. Native PAGE can also be used to study protein-protein interactions and to purify proteins.

[0033] To perform native PAGE, a gel is made from polyacrylamide, a cross-linked polymer that forms a mesh. The gel is then placed in an electrophoresis chamber and filled with a buffer solution. The proteins to be separated are loaded onto the gel and an electric field is applied. The proteins migrate through the gel at different speeds depending on their size, charge, and shape. Smaller proteins move faster through the gel than larger proteins, and proteins with a higher charge move faster than proteins with a lower charge.

[0034] After the proteins have been separated, the gel can be stained to visualize the bands. A common stain used for native PAGE is Coomassie blue, which stains all proteins blue. Once the gel has been stained, the proteins can be identified and their native conformation can be studied.

[0035] Initially, at step 102, one or more blood samples are collected from at least one patient without mixing with Ethylenediaminetetraacetic acid (EDTA) to avoid any interference with a zymogram.

[0036] At step 104, a plasma is separated from the blood sample by centrifuging the blood samples at 5000 rpm for at least 20 min in order to obtain extracellular enzymes. At step 106, the separated plasma is then transferred into conduit without disturbing a cell pellet upon centrifugation and the conduit filled with plasma is stored in an ice container.

[0037] At step 108, a pair of gel samples containing 12 weight percentage of separating gel and 5 weight percentage of stacking gel are prepared using a gel electrophoresis unit and then the pair of gel samples are solidified to attain solidified gel samples. The separating gel and stacking gel contains a gelatin of about 1 mg/ml.

[0038] At step 110, the solidified gel samples are loaded with equal volume of plasma mixed with a native gel loading dye solution upon solidification and then the solidified gel samples are loaded into wells for performing a native PAGE (polyacrylamide gel electrophoresis). The native PAGE (polyacrylamide gel electrophoresis) is performed in the 1X Tris –Glycine buffer at 120 volts for a time period of at least 1 hour.

[0039] At step 112, the solidified gel samples are removed from the wells without any tearing and the solidified gel is incubated in a buffer solution for a time period of 24 hours at a temperature of about 37 degrees. The buffer solution used is about a volume 50 ml, which contains 2.5 ml of 50 mM Tris Hcl, 125 µl of 5 mM Cacl2, 0.1 M and 0.5 µl of Zncl2.

[0040] At step 114, the incubated gel from the buffer solution is removed and rinsed gently with deionized water for removing any remnants of the buffer solution. Further at step 116, the incubated gel is stained for at least one day overnight at a room temperature of 37 degrees for obtaining a stained gel and the stained gel is rinsed with deionized water to remove at least one dye until clearance zones around one or more protein bands are observed clearly. The at least one dye includes a coomassie brilliant blue dye.

[0041] According to another exemplary embodiment of the invention, FIG. 2A refers to a panel representing a control Native Zymo performed using a purified enzyme. The panel show the clearance of band in the form of dark blue color indicating the enzyme activity.

[0042] According to another exemplary embodiment of the invention, FIG. 2B refers to a control Native Zymo performed using healthy volunteer plasma and patient plasma. The panel as shown in the FIG. 2B shows the comparison of healthy volunteer plasma with patients plasma effected with atherosclerosis clearance zones are shown prominently for the patients samples rather than the healthy volunteer samples. The zymography method detects active molecules of the bacterial or plant or mammalian cell proteasomes without interrupting the zymogen in the protein structure.

[0043] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure a method for a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis process with zymography, is disclosed. The proposed invention provides a method for estimating enzyme activity by combining native polyacrylamide gel electrophoresis process with zymography, which helps in rapid detection of enzyme activity of various proteinases without lengthy procedures of enzyme extraction, purification and evaluation.

[0044] The proposed invention provides a method for estimating enzyme activity that does not disturb a cysteine switch while activating MMPs (matrix metalloproteinases) and makes detection of enzymes easier. The method overcomes the challenges like extraction, purification and evaluation of enzymes. The method helps in detecting active molecules of the bacterial or plant or mammalian cell proteasomes without interrupting the zymogen in the protein structure. The method improves detection capabilities of malfunctioning enzymes in clinical settings and enhances the pace of biomedical research. The method is highly beneficial for testing the unpurified samples such as blood-plasma.

[0045] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application
,CLAIMS:CLAIMS:
I/We Claim:
1. A method for estimating enzyme activity by combining a native polyacrylamide gel electrophoresis process with zymography, comprising:
collecting blood samples from at least one patient without mixing the blood sample with Ethylenediaminetetraacetic acid (EDTA) to avoid any interference with a zymogram;
separating a plasma from the blood sample by centrifuging the blood samples at 5000 rpm for at least 20 min in order to obtain extracellular enzymes;
transferring the separated plasma into a conduit without disturbing a cell pellet upon centrifugation and storing the conduit filled with plasma in an ice container;
preparing a pair of gel samples containing 12 weight percentage of separating gel and 5 weight percentage of stacking gel using a gel electrophoresis unit and then solidifying the pair of gel samples to attain solidified gel samples;
loading the solidified gel samples with equal volumes of plasma and a native gel loading dye solution upon solidification and loading the solidified gel samples into wells for performing a native PAGE (polyacrylamide gel electrophoresis);
removing the solidified gel from the wells without any tearing and incubating the solidified gel in a buffer solution for a time period of 24 hours at a temperature of about 37°C;
removing the incubated gel from the buffer solution and rinsing the incubated gel gently with deionized water for removing any remnants of the buffer solution; and
staining the incubated gel with at least one dye overnight at a room temperature for obtaining a stained gel and the stained gel is rinsed with deionized water to remove at least one dye until clearance zones around a one or more protein bands are observed clearly.
2. The method as claimed in claim 1, wherein the separating gel and stacking gel contains a gelatin of about 1 mg/ml.
3. The method as claimed in claim 1, wherein the buffer solution used is about a volume 50 ml, which contains 2.5 ml of 50 mM Tris Hcl, 125 µl of 5 mM Cacl2, 0.1 M and 0.5 µl of Zncl2.
4. The method as claimed in claim 1, wherein the at least one dye includes a coomassie brilliant blue dye.
5. The method as claimed in claim 1, wherein the native PAGE (polyacrylamide gel electrophoresis) is performed in the 1X Tris –Glycine buffer at 120 volts for a time period of at least 1 hour.