methods of enzyme assay
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The document outlines various methods for assaying enzyme activity, emphasizing the significance of enzyme assays in fields such as enzyme kinetics and inhibition. It details continuous and discontinuous assay types, various detection methods (spectrophotometric, fluorescence, radioisotopic, chemiluminescent, etc.), and immunochemical techniques such as enzyme immunoassays. Ultimately, these assays enable the measurement of enzyme activity and have broad applications in biochemistry and diagnostics.
![At low concentrations, the intensity of fluorescence (fr) is related to the intensity of the incident
light (fo) of appropriate wavelength by the relationship:
Where is the molar absorption coefficient, c the molar concentration, l the length of the light-path
and q the quantum efficiency (i.e. the number of quanta fluoresced divided by the number of
quanta absorbed)
An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH.
Here, the reduced forms are fluorescent and the oxidised forms nonfluorescent.
Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction
reactions by an increase.
More sensitive than spectrophotometric assays, but can suffer from interference caused by
impurities and the instability of many fluorescent compounds when exposed to light.
Detection in small quantities
Non dangerous
Other example:-
RADIOISOTOPIC METHOD:
• The use of a radioactively-labelled substrate can be valuable in enzymatic analysis. The
isotopes most commonly used for labelling purposes are 3H (tritium), 14C(carbon),
32P(phosphorous), 35S(sulphur) and 131I(iodine). All of these isotopes emit beta-radiation
(electrons) as they decay.
• After the enzyme-catalysed reaction has progressed for a specified period, it is terminated.
The substrate is then separated from the product, usually by chromatography or
electrophoresis, and the product concentration is determined indirectly by measuring the
radioactivity of the product fraction.
• A typical example of enzymatic analysis by a radiochemical procedure is that involving the
cholinesterase-catalysed hydrolysis of [ 14C]-acetylcholine.](https://image.slidesharecdn.com/enzymeassay1-180329151646/85/methods-of-enzyme-assay-5-320.jpg)
methods of enzyme assay
- 1. Methods to assay enzymes Introduction: • Assay is an act of analyzing test or appraisal to determine the components of a substance or object. • Enzyme assays are laboratory methods for measuring enzymatic activity. • They are vital for the study of enzyme kinetics and enzyme inhibition. • The assay is the act of measuring how fast a given (unknown) amount of enzyme will convert substrate to product (the act of measuring a velocity). • Enzyme assays measure either the disappearance of substrate over time or the appearance of product over time. Types of Enzyme assay Two types (Based on sampling methods) 1. Continuous assays Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. It gives a continuous reading of activity, multiple measurements, usually of absorbance change are made during the reaction either at specific time intervals (usually every 30 or 60 seconds) or continuously by a continuous- recording spectrophotometer. A few methods are spectrophotoometric, fluorometric, calorimetric and chemi- luminescent. 2. Discontinuous assays: In this assay where the samples are taken, the reaction stopped and then the concentration of substrates/products determined. The discontinuous assays are radiometric and chromatographic.
- 2. Features of a good Enzyme assay • Simple and Specific • Rapid (one doesn’t need to wait for hrs or weeks for the results to appear) • Sensitive ( very little sample) • Easy to use • Economical Measurement of enzyme activity by spectroscopy • The spectrophotometric assay is the most common method of detection in enzyme assays. • The assay uses a spectrophotometer, a machine used to measure the amount of light a substance's absorbs, to combine kinetic measurements and Beer's law by calculating the appearance of product or disappearance of substrate concentrations. • The spectrophotometric assay is simple, non-destructive, selective, and sensitive. The following spectroscopic techniques are used: Fluorescence spectroscopy, UV/VIS Spectroscopy, Spectrophotometric Assays, and Infrared spectroscopy. UV/VIS SPECTROSCOPY: UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400 nm.) is absorbed by the molecule. Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground state to higher energy state. The energy of the ultra-violet radiation that are absorbed is equal to the energy difference between the ground state and higher energy states (deltaE = hf). Principle of UV spectroscopy UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution. The expression of Beer-Lambert law is- A = log (I0/I) = Ecl Where, A = absorbance I0 = intensity of light incident upon sample cell I = intensity of light leaving sample cell
- 3. C = molar concentration of solute L = length of sample cell (cm.) E = molar absorptivity From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing light of a given wavelength, the greater the extent of light absorption. This is the basic principle of UV spectroscopy. Instrumentation and working of UV spectroscopy Instrumentation and working of the UV spectrometers can be studied simultaneously. Most of the modern UV spectrometers consist of the following parts- Light Source- Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Monochromator- Monochromators generally composed of prisms and slits. The various wavelengths of the light source which are separated by the prism are then selected by the slits. Sample and reference cells- One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. Detector- Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.
- 4. Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound. UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidized forms. Direct versus coupled assays Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase. Here, the product of one reaction is used as the substrate of another, easily detectable reaction. For example, figure shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase. FLURESCENCE METHOD/FLUORIMETRIC: Compounds are said to be fluorescent when they absorb light of one wavelength and then emit light of a longer wavelength.
- 5. At low concentrations, the intensity of fluorescence (fr) is related to the intensity of the incident light (fo) of appropriate wavelength by the relationship: Where is the molar absorption coefficient, c the molar concentration, l the length of the light-path and q the quantum efficiency (i.e. the number of quanta fluoresced divided by the number of quanta absorbed) An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms nonfluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase. More sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light. Detection in small quantities Non dangerous Other example:- RADIOISOTOPIC METHOD: • The use of a radioactively-labelled substrate can be valuable in enzymatic analysis. The isotopes most commonly used for labelling purposes are 3H (tritium), 14C(carbon), 32P(phosphorous), 35S(sulphur) and 131I(iodine). All of these isotopes emit beta-radiation (electrons) as they decay. • After the enzyme-catalysed reaction has progressed for a specified period, it is terminated. The substrate is then separated from the product, usually by chromatography or electrophoresis, and the product concentration is determined indirectly by measuring the radioactivity of the product fraction. • A typical example of enzymatic analysis by a radiochemical procedure is that involving the cholinesterase-catalysed hydrolysis of [ 14C]-acetylcholine.
- 6. • Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. • They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells). • Radioactivity is usually measured in these procedures using a scintillation counter., which measures the ionizing radiation. • Very sensitive but hazardous CHEMILUMINESCENT: It is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected. ION SELECTIVE ELECTRODE: An ion-selective electrode (ISE), also known as a specific ion electrode (SIE), is a transducer (or sensor) that converts the activity of a specific ion dissolved in a solution into an electrical potential. The voltage is theoretically dependent on the logarithm of the ionic activity, according to the Nernst equation. Ion-selective electrodes are used in analytical chemistry and biochemical/biophysical research, where measurements of ionic concentration in an aqueous solution are required. Working Mechanism • The ion-selective electrode works based on the principle of a galvanic cell. It consists of a reference electrode, ion-selective membrane and voltmeter. • The transport of ions from an area of high concentration to low concentration through the selective binding of ions with the specific sites of the membrane creates a potential difference. • This potential is measured with respect to a stable reference electrode having a constant potential, and a net charge is determined. The difference in potential between the electrode and the membrane depends on the activity of the specific ion in solution. • The strength of the net charge thus measured is directly proportional to the concentration of the selected ion.
- 7. • The electric potential can be calibrated by direct means, standard additions and titrations. However, direct calibration is the most common means of measuring concentrations. Enzyme electrodes definitely are not true ion-selective electrodes but usually are considered within the ion-specific electrode topic. Such an electrode has a "double reaction" mechanism - an enzyme reacts with a specific substance, and the product of this reaction (usually H+ or OH−) is detected by a true ion-selective electrode, such as a pH-selective electrodes. All these reactions occur inside a special membrane which covers the true ion-selective electrode, which is why enzyme electrodes sometimes are considered as ion-selective. An example is glucose selective electrodes Advanteages - Exhibit wide response - Exhibit wide linear range - Low cost - Color or turbidity of analyte does not affect results - Come in different shapes and sizes OXYGEN ELECTRODE The oxygen electrode is an electrode that measures ambient oxygen concentration in a liquid using a catalytic platinum surface according to the net reaction: O2 + 4 e− + 4 H+ → 2 H2O It improves on a bare platinum electrode by use of a membrane to reduce fouling and metal plating onto the platinum.
- 8. Mechanism of Oxygen (pO2) Electrode: The oxygen electrode laid the basis for the first glucose biosensor (in fact the first biosensor of any type), invented by Clark and Lyons in 1962. The basic concept of the glucose biosensor is based on the fact that the immobilized glucose oxidase (GOx) catalyzes the oxidation of β-D-glucose by molecular oxygen producing gluconic acid and hydrogen peroxide. In order to work as a catalyst, GOx requires a redox cofactor—flavin adenine dinucleotide (FAD). FAD works as the initial electron acceptor and is reduced to FADH2. Glucose + GOx − FAD+ → Glucolactone + GOx − FADH2 The cofactor is regenerated by reacting with oxygen, leading to the formation of hydrogen peroxides. GOx − FADH2 + O2 → GOx − FAD + H2 O2 Hydrogen peroxide is oxidized at a catalytic, classically platinum (Pt) anode. The electrode easily recognizes the number of electron transfers, and this electron flow is proportional to the number of glucose molecules present in blood. H2O2 → 2H+ + O2 + 2e IMMUNOCHEMICAL METHODS Enzyme immunoassays utilize enzymes, usually peroxidase or alkaline phosphatase, to detect and quantify immunochemical reactions. Both antibodies or antigens can be labelled with an enzyme in order to aid detection. Types: • Heterogeneous enzyme immunoassay • Homogeneous enzyme immunoassay Heterogeneous enzyme immunoassay:
- 9. A heterogeneous enzyme immunoassay method is also called enzyme-linked immunosorbent assay (ELISA). In this type of assay, one of the immunochemical reaction components (antigen or antibody) is first non-specifically adsorbed to the surface of a solid phase. Tubes, wells of microtiter plates, and magnetic particles may be used as the solid phases. The solid phase facilitates separation of bound- and free-labelled reactants. Homogeneous enzyme immunoassay: A homogeneous enzyme immunoassay is a sort of enzyme multiplied immunoassay technique (EMIT) that does not require a separation of bound and free labelled antibodies or antigens. It is simple to perform and has been used for estimation of drugs, hormones and metabolites. Sample containing the estimated antigen is mixed with a known quantity of the same antigen labelled with enzyme (conjugate); and limited amount of specific antibody is added. The unlabelled antigen from the sample competes with the conjugate for the antibody. Binding of antibody on the conjugate results in loss of enzyme activity due to blocking the enzyme active site or change of its conformation. The more unlabelled antigen is present in the solution, the less conjugate will bind to the antibody, and more enzyme activity will be preserved in the solution. Therefore, the enzyme activity is proportional to the antigen concentration in the sample. The reaction scheme in these immunochemical assays can follow either competitive, or non- competitive approach. Competitive enzyme immunoassay: This assay is always performed under condition of antigen excess. The enzyme-labelled antigen (conjugate) is mixed with serum sample containing the unknown amount of antigen. The serum antigen and enzyme-labelled antigen compete for binding sites of a limited quantity of specific antibodies bound to the solid phase. Labelled and non-labelled antigen bind to the antibody in the same proportion as is their proportion in the reaction mixture. In other words, the more non- labelled antigen is contained in the mixture the less labelled antigen is bound.
- 10. Principle of competitive immunoassays – various proportions of antigen and antibody Under these conditions the probability of the antibody binding the labelled antigen is inversely proportional to the concentration of unlabelled antigen. The higher the amount of unlabelled antigen in the sample, the more labelled antigen remains free (unbound). After an incubation, all the unbound both enzyme-labelled and unlabelled antigens are removed by washing along with all other serum constituents. In the subsequent indicator reaction for detection of enzyme activity a chromogenic substrate for the enzyme label is added. The intensity of colour is inversely proportional to the concentration of the antigen in serum sample. The results are obtained from a calibration curve constructed with the standards of known concentration of antigen
- 11. Competitive enzyme immunoassay Non-competitive enzyme immunoassay (sandwich methods): This kind of enzyme immunoassay can be adopted for measurement of either antigens or antibodies. It is a heterogeneous immunoassay using a solid phase coated with antibody or antigen, which must always be in excess over the analyte being measured. Non-competitive enzyme immunoassay for determination of antigen: This non-competitive enzyme immunoassay is suitable for the measurement of large antigens with several antibody-binding sites. Two different molecules of antibodies directed against various epitopes are necessary for performing the assay. The first antibody is in excess adsorbed to a solid phase. The serum sample or calibrators containing the desired antigen are added to the well with immobilised antibody. The first imunochemical reaction occurs. Since the antibody is in excess, all antigen molecules should bind. After an incubation, all the non-reacting material in the sample is washed away. Then a second enzyme-labelled antibody (different from the first antibody) is added in excess. In the second immunochemical reaction, another antigen epitope binds to the second labelled antibody. “Sandwich complex“ consisting of solid-phase antibody – antigen – enzymelabelled antibody is formed. After washing of all the unreacted enzyme-labelled antibody,
- 12. the substrate is added. The intensity of the finally measured coloured product of the enzyme reaction is directly proportional to the amount of antigen. Non-competitive enzyme immunoassay for determination of antigen Example of fast immunochemical diagnostic methods Detection of human chorionic gonadotropin (pregnancy test) Porous membrane that serves as a support for the test is divided into four areas. Three different antibodies are employed. The first sample area contains specific anti-hCG antibody labelled with microparticles of colloid gold or blue latex (Ab1). When urine sample is applied molecules of this antibody flow to the second, detection area. Another specific antibody against hCG (Ab2) is anchored in this area. If chorionic gonadotropin is contained in the sample a combined immunocomplex with both labelled and anchored antibody is formed (Ab2-hCG-Ab1) and a coloured band displays in the detection area. Excess of the labelled antibody is caught in the third (control) area with immobilised antibodies
- 13. against labelled anti-hCG. A band in the control area is formed, indicating that the test works properly. Thus, two bands are interpreted as a positive result. In case the urine sample contains no hCG the labelled antibody is bound in the third area only. One band in the control zone only is therefore read as a negative result. If no band is displayed in the control area the test is invalid. Some tests are so designed that the control zone has a shape of a minus sign and the detection zone crosses it perpendicularly. A plus sign therefore appears in case of positive result. Tentative test for hCG in urine