Zymoblot
Encyclopedia
Zymoblot is the fastest available microtechnique to detect gene expression
or enzyme activity in any biological specimen. The technique was invented by Professor Elsayed Elsayed Wagih
in collaboration with Professor Jacqueline Fletcher of the Department of Plant Pathology, Noble Research Centre, Oklahoma State University, USA in 1993.
activities in living organisms is therefore one of the most commonly performed activities in modern physiology
laboratories. Numerous methods of enzyme assay
s are available to quantitatively follow enzyme reactions. These methods which have been grouped in six categories, namely, spectrophotometric, fluorescence
, nanometric, electrode
, polarimetric, radiobiochemical are with no drawbacks. Recently, a new qualitative or rather semi quantitative micro-technique, first described by Wagih and Fletcher (1993), and termed 'zymoblot' has been introduced to detect enzyme activities in spiroplasma
s and bacteria
and many other biological system
s. Later, the technique was made quantitative by densitometry
and successfully used to monitor peroxidase
activity in virus
infected plants.
, may require larger aliquot
s of a sample so that the amount of the coloured soluble products produced is large enough to colour the content of the assay cuvette
to a colorimetrically readable level. The technique has advantages not shared by any other technique. Samples to be analysed by zymoblot require no dialysis (a process that may take days) as washing blots in Tris-buffered saline
(TBS) before marinating them in the reaction mixture does remove inhibitor
s. In contrast to the other techniques where samples are assayed individually, samples to be analysed by zymoblot are spotted on the same blot and enzyme activity is assayed with the same reaction mixture at the same time minimising experimental errors and allowing quick qualitative comparisons. Additionally, several enzymes can be assayed in a sequential order on the same blot. That is to say, if a blot proves negative for a certain enzyme, it can be washed in TBS and reused for another enzyme and so on until a positive reaction for an enzyme is obtained. Unlike wet assays (e.g. colorimetry), results obtained by the zymoblot are always in a recorded from. This allows zymoblots to be carried out in one place, where a densitometer
may not be available, and taken or sent to another place to be quantitatively assayed by densitometry. Taking a zymoblot in a researcher's wallet to a meeting facilitates discussions and exchange of ideas with other scientists. While immunologically
-based enzyme assays which uses enzyme specific antibodies
to directly detect enzymes suffers from the major disadvantage of measuring the "total enzyme content" and not the total enzyme activity, zymoblot, being not a serological technique
, uses no antibodies and measures enzyme activity as it detects only the active (functional) portion of the total enzyme content in a sample. Zymoblot is an end-point type of assay where an enzyme reaction is allowed to proceed for a fixed period of time before being stopped by rapid elimination of its specific substrate
. However, the technique could be adapted for the continuous enzyme assay when colour intensity is monitored over time by incubating sister blots for progressively increasing periods of time. When different samples are compared on the same blot, the reaction should be stopped sometime during the linear part of the course of the reaction. This could be judged upon visually and the reaction is stopped when differences in colour intensity among spots are evident, taking into consideration that the duration of linearity in some enzyme reactions may be very short indeed.
, agriculture
and industrial biotechnology
and, more broadly, in general biotechnology
application. It is useful in studies including physiology of humans, animals, plants and microorganisms, differential diagnosis
of diseases and identification of pathogen
s, biotaxonomy of organisms, stress and pathogenesis
physiology, physiological basis for disease resistance, developmental physiology and screening for commercially important enzymes and many other applications. The technique is particularly useful when initial testing for enzyme activity is required. It can be used in investigations involving screening a living organism for large numbers of enzymes. It can also be very handy in studying enzyme distribution or tissue-specific gene expression in terms of enzyme activity throughout the body or across an organ
. Samples taken from different parts of an organism or an organ can simultaneously be analysed and compared, on the same blot, giving a clear picture of the distribution of enzyme activity. With the same simplicity, corresponding tissues taken from normally and biotically- or abiotically-stressed individuals can be compared for enzyme stimulation or induction. Furthermore, zymoblots can be very helpful in cytochemodissection studies aiming at localising enzymes within cells. Cell fractions representing different parts of the cell (nuclei
, mitochondria, lysosome
s, peroxisome
s, Golgi bodies, cytosol
,... etc.) can be tested for a host of enzymes in a relatively short time.
Qualitative zymoblot is of great potential use in diagnosis of human, animal and plant disease
s. If a pathogen demonstrates a specific enzyme that is not shared by its host, the technique can be a definitive diagnostic tool. The detection of an enzyme not known to be normally present in a sample of body fluid (e.g. blood serum
, CSF
, synovial fluid
, milk, tears…etc.) using qualitative zymoblot is an indication of a physiological disorder, inflammatory reaction or pathogenic infection. In all these situations, quantitative zymoblot can be used to determine the severity of the problem. Similarly, replacing biological fluids with non-biological fluids taken from water bodies (or prepared from similar environmental sources) at different locations or depths at different times will allow zymoblot, through detecting enzyme activities, to reveal or monitor microbial load and consequently determine the level of contamination in these sources.
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
or enzyme activity in any biological specimen. The technique was invented by Professor Elsayed Elsayed Wagih
Elsayed Elsayed Wagih
Elasayed Elsayed Wagih PhD, DIC, CIDTT is an Egyptian Professor of Virology and Biotechnology and vice President of the Arab Society for Biotechnology. He was born in Alexandria, Egypt...
in collaboration with Professor Jacqueline Fletcher of the Department of Plant Pathology, Noble Research Centre, Oklahoma State University, USA in 1993.
Background
Physiological phenomena whether at the cellular or molecular level in living organisms are driven either directly or indirectly by enzyme reactions. The assay of enzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
activities in living organisms is therefore one of the most commonly performed activities in modern physiology
Physiology
Physiology is the science of the function of living systems. This includes how organisms, organ systems, organs, cells, and bio-molecules carry out the chemical or physical functions that exist in a living system. The highest honor awarded in physiology is the Nobel Prize in Physiology or...
laboratories. Numerous methods of enzyme assay
Enzyme assay
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.-Enzyme units:...
s are available to quantitatively follow enzyme reactions. These methods which have been grouped in six categories, namely, spectrophotometric, fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
, nanometric, electrode
Electrode
An electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit...
, polarimetric, radiobiochemical are with no drawbacks. Recently, a new qualitative or rather semi quantitative micro-technique, first described by Wagih and Fletcher (1993), and termed 'zymoblot' has been introduced to detect enzyme activities in spiroplasma
Spiroplasma
Spiroplasma is a genus of Mollicutes, a group of small bacteria without cell walls. Spiroplasma shares the simple metabolism, parasitic lifestyle, fried-egg colony morphology and small genome of other Mollicutes, but has a distinctive helical morphology, unlike Mycoplasma. It has a spiral shape...
s and bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
and many other biological system
Biological system
In biology, a biological system is a group of organs that work together to perform a certain task. Common systems, such as those present in mammals and other animals, seen in human anatomy, are those such as the circulatory system, the respiratory system, the nervous system, etc.A group of systems...
s. Later, the technique was made quantitative by densitometry
Densitometry
Densitometry is the quantitative measurement of optical density in light-sensitive materials, such as photographic paper or film, due to exposure to light...
and successfully used to monitor peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
activity in virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
infected plants.
Procedure
As little as 1µl, or less, of a sample is enough to detect enzyme activity by the zymoblot technique as the coloured product being insoluble, accumulates at a confined area over the spotting site. The other techniques, based on colorimetryColorimetry
Colorimetry is "the science and technology used to quantify and describe physically the human color perception."It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color perception, most often the CIE 1931 XYZ color space...
, may require larger aliquot
Aliquot
Aliquot may refer to:In mathematics:*Aliquot part, a proper divisor of an integer*Aliquot sum, the sum of the aliquot parts of an integer*Aliquot sequence, a sequence of integers in which each number is the aliquot sum of the previous numberIn music:...
s of a sample so that the amount of the coloured soluble products produced is large enough to colour the content of the assay cuvette
Cuvette
A cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz and designed to hold samples for spectroscopic experiments. The best cuvettes are as clear as possible, without impurities that might affect a spectroscopic reading...
to a colorimetrically readable level. The technique has advantages not shared by any other technique. Samples to be analysed by zymoblot require no dialysis (a process that may take days) as washing blots in Tris-buffered saline
Tris-Buffered Saline
Tris-buffered saline is a buffer used in some biochemical techniques to maintain the pH within a relatively narrow range. Tris has a slightly alkaline buffering capacity in the 7–9.2 range....
(TBS) before marinating them in the reaction mixture does remove inhibitor
Inhibitor
Something that restrains, blocks, or suppresses.Inhibitor or inhibition may refer to:* Corrosion inhibitor, a substance that decreases the rate of metal oxidation...
s. In contrast to the other techniques where samples are assayed individually, samples to be analysed by zymoblot are spotted on the same blot and enzyme activity is assayed with the same reaction mixture at the same time minimising experimental errors and allowing quick qualitative comparisons. Additionally, several enzymes can be assayed in a sequential order on the same blot. That is to say, if a blot proves negative for a certain enzyme, it can be washed in TBS and reused for another enzyme and so on until a positive reaction for an enzyme is obtained. Unlike wet assays (e.g. colorimetry), results obtained by the zymoblot are always in a recorded from. This allows zymoblots to be carried out in one place, where a densitometer
Densitometer
A densitometer is a device that measures the degree of darkness of a photographic or semitransparent material or of a reflecting surface. The densitometer is basically a light source aimed at a photoelectric cell. It determines the density of a sample placed between the light source and the...
may not be available, and taken or sent to another place to be quantitatively assayed by densitometry. Taking a zymoblot in a researcher's wallet to a meeting facilitates discussions and exchange of ideas with other scientists. While immunologically
Immunology
Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. It deals with the physiological functioning of the immune system in states of both health and diseases; malfunctions of the immune system in immunological disorders ; the...
-based enzyme assays which uses enzyme specific antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
to directly detect enzymes suffers from the major disadvantage of measuring the "total enzyme content" and not the total enzyme activity, zymoblot, being not a serological technique
Serology
Serology is the scientific study of blood serum and other bodily fluids. In practice, the term usually refers to the diagnostic identification of antibodies in the serum...
, uses no antibodies and measures enzyme activity as it detects only the active (functional) portion of the total enzyme content in a sample. Zymoblot is an end-point type of assay where an enzyme reaction is allowed to proceed for a fixed period of time before being stopped by rapid elimination of its specific substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
. However, the technique could be adapted for the continuous enzyme assay when colour intensity is monitored over time by incubating sister blots for progressively increasing periods of time. When different samples are compared on the same blot, the reaction should be stopped sometime during the linear part of the course of the reaction. This could be judged upon visually and the reaction is stopped when differences in colour intensity among spots are evident, taking into consideration that the duration of linearity in some enzyme reactions may be very short indeed.
Applications
The Zymoblot technique is simpler, cheaper, more reliable and less time consuming than all known procedures for enzyme assays. It is probably the quickest available technique to detect enzyme activity in any biological or even non-biological specimen. The technique is highly competitive in price with all commercially available kits. Such advantages should qualify the Zymoblot technique for wide potential uses in medicineMedicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
, agriculture
Agriculture
Agriculture is the cultivation of animals, plants, fungi and other life forms for food, fiber, and other products used to sustain life. Agriculture was the key implement in the rise of sedentary human civilization, whereby farming of domesticated species created food surpluses that nurtured the...
and industrial biotechnology
Industrial biotechnology
Industrial biotechnology is the application of biotechnology for industrial purposes, including manufacturing, alternative energy , and biomaterials. It includes the practice of using cells or components of cells like enzymes to generate industrially useful products...
and, more broadly, in general biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
application. It is useful in studies including physiology of humans, animals, plants and microorganisms, differential diagnosis
Differential diagnosis
A differential diagnosis is a systematic diagnostic method used to identify the presence of an entity where multiple alternatives are possible , and may also refer to any of the included candidate alternatives A differential diagnosis (sometimes abbreviated DDx, ddx, DD, D/Dx, or ΔΔ) is a...
of diseases and identification of pathogen
Pathogen
A pathogen gignomai "I give birth to") or infectious agent — colloquially, a germ — is a microbe or microorganism such as a virus, bacterium, prion, or fungus that causes disease in its animal or plant host...
s, biotaxonomy of organisms, stress and pathogenesis
Pathogenesis
The pathogenesis of a disease is the mechanism by which the disease is caused. The term can also be used to describe the origin and development of the disease and whether it is acute, chronic or recurrent...
physiology, physiological basis for disease resistance, developmental physiology and screening for commercially important enzymes and many other applications. The technique is particularly useful when initial testing for enzyme activity is required. It can be used in investigations involving screening a living organism for large numbers of enzymes. It can also be very handy in studying enzyme distribution or tissue-specific gene expression in terms of enzyme activity throughout the body or across an organ
Organ (anatomy)
In biology, an organ is a collection of tissues joined in structural unit to serve a common function. Usually there is a main tissue and sporadic tissues . The main tissue is the one that is unique for the specific organ. For example, main tissue in the heart is the myocardium, while sporadic are...
. Samples taken from different parts of an organism or an organ can simultaneously be analysed and compared, on the same blot, giving a clear picture of the distribution of enzyme activity. With the same simplicity, corresponding tissues taken from normally and biotically- or abiotically-stressed individuals can be compared for enzyme stimulation or induction. Furthermore, zymoblots can be very helpful in cytochemodissection studies aiming at localising enzymes within cells. Cell fractions representing different parts of the cell (nuclei
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
, mitochondria, lysosome
Lysosome
thumb|350px|Schematic of typical animal cell, showing subcellular components. [[Organelle]]s: [[nucleoli]] [[cell nucleus|nucleus]] [[ribosomes]] [[vesicle |vesicle]] rough [[endoplasmic reticulum]]...
s, peroxisome
Peroxisome
Peroxisomes are organelles found in virtually all eukaryotic cells. They are involved in the catabolism of very long chain fatty acids, branched chain fatty acids, D-amino acids, polyamines, and biosynthesis of plasmalogens, etherphospholipids critical for the normal function of mammalian brains...
s, Golgi bodies, cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
,... etc.) can be tested for a host of enzymes in a relatively short time.
Qualitative zymoblot is of great potential use in diagnosis of human, animal and plant disease
Disease
A disease is an abnormal condition affecting the body of an organism. It is often construed to be a medical condition associated with specific symptoms and signs. It may be caused by external factors, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune...
s. If a pathogen demonstrates a specific enzyme that is not shared by its host, the technique can be a definitive diagnostic tool. The detection of an enzyme not known to be normally present in a sample of body fluid (e.g. blood serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
, CSF
Cerebrospinal fluid
Cerebrospinal fluid , Liquor cerebrospinalis, is a clear, colorless, bodily fluid, that occupies the subarachnoid space and the ventricular system around and inside the brain and spinal cord...
, synovial fluid
Synovial fluid
Synovial fluid is a viscous, non-Newtonian fluid found in the cavities of synovial joints. With its yolk-like consistency , the principal role of synovial fluid is to reduce friction between the articular cartilage of synovial joints during movement.-Overview:The inner membrane of synovial joints...
, milk, tears…etc.) using qualitative zymoblot is an indication of a physiological disorder, inflammatory reaction or pathogenic infection. In all these situations, quantitative zymoblot can be used to determine the severity of the problem. Similarly, replacing biological fluids with non-biological fluids taken from water bodies (or prepared from similar environmental sources) at different locations or depths at different times will allow zymoblot, through detecting enzyme activities, to reveal or monitor microbial load and consequently determine the level of contamination in these sources.