![DU640 spectrophotometer](https://upload.wikimedia.org/wikipedia/commons/e/ed/DU640_spectrophotometer.jpg)
Enzyme assays are
laboratory
A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...
methods for measuring
enzymatic
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
activity. They are vital for the study of
enzyme kinetics
Enzyme kinetics is the study of the rates of enzyme-catalysed chemical reactions. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in th ...
and
enzyme inhibition
An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a sp ...
.
Enzyme units
The quantity or concentration of an enzyme can be expressed in
molar amounts, as with any other chemical, or in terms of activity in
enzyme unit The enzyme unit, or international unit for enzyme (symbol U, sometimes also IU) is a unit of enzyme's catalytic activity.
1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per mi ...
s.
Enzyme activity
Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on various physical conditions, ''which should be specified''.
It is calculated using the following formula:
:
where
:
Enzyme activity
:
Moles of substrate converted per unit time
:
Rate of the reaction
:
Reaction volume
The SI unit is the
katal
The katal (symbol: kat) is the unit of catalytic activity in the International System of Units (SI) used for quantifying the catalytic activity of enzymes (that is, measuring the enzymatic activity level in enzyme catalysis) and other catalys ...
, 1 katal = 1
mol s
−1 (mole per second), but this is an excessively large unit. A more practical and commonly used value is
enzyme unit The enzyme unit, or international unit for enzyme (symbol U, sometimes also IU) is a unit of enzyme's catalytic activity.
1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per mi ...
(U) = 1
μmol min
−1 (micromole per minute). 1 U corresponds to 16.67
nanokatals.
Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as
gelatin
Gelatin or gelatine (from la, gelatus meaning "stiff" or "frozen") is a translucent, colorless, flavorless food ingredient, commonly derived from collagen taken from animal body parts. It is brittle when dry and rubbery when moist. It may also ...
, then measured in ''gelatin digesting units'' (GDU), or milk proteins, then measured in ''milk clotting units'' (MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU approximately equals 1.5 MCU.
An increased amount of substrate will increase the rate of reaction with enzymes, however once past a certain point, the rate of reaction will level out because the amount of active sites available has stayed constant.
Specific activity
The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min
−1 mg
−1). Specific activity gives a measurement of enzyme purity in the mixture. It is the micro moles of product formed by an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
in a given amount of time (minutes) under given conditions per milligram of total proteins. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal/kg, but a more practical unit is μmol/mgmin.
Specific activity is a measure of ''enzyme processivity'' (the capability of enzyme to be processed), at a specific (usually saturating)
substrate concentration, and is usually constant for a pure enzyme.
An active site titration process can be done for the elimination of errors arising from differences in cultivation batches and/or misfolded enzyme and similar issues. This is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible inhibitor. The specific activity should then be expressed as μmol min
−1 mg
−1 active enzyme. If the molecular weight of the enzyme is known, the
turnover number Turnover number has two different meanings:
In enzymology, turnover number (also termed ''k''cat) is defined as the maximum number of chemical conversions of substrate molecules per second that a single active site will execute for a given enzyme ...
, or μmol product per second per μmol of active enzyme, can be calculated from the specific activity. The turnover number can be visualized as the number of times each enzyme molecule carries out its catalytic cycle per second.
Related terminology
The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L
−1 s
−1).
The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated and then getting a clear result.
Types of assays
All enzyme
assay
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...
s measure either the consumption of substrate or production of product over time.
A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:
* Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient.
Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation that the amount of free substrate is approximately equal to the amount of the initial substrate can be made.
The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.
* Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. Progress curve experiments were widely used in the early period of enzyme kinetics, but are less common now.
* Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require specialist techniques (such as
flash photolysis
Flash photolysis is a pump-probe laboratory technique, in which a sample is first excited by a strong pulse of light from a pulsed laser of nanosecond, picosecond, or femtosecond pulse width or by another short-pulse light source such as a fla ...
of caged compounds) or rapid mixing (such as
stopped-flow Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms, introduced by Britton Chance and extended by Quentin Gibson (Other techniques, such as the temperature-jump method, are available for m ...
, quenched flow or continuous flow).
* Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a
temperature
Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer.
Thermometers are calibrated in various temperature scales that historically have relied o ...
,
pressure
Pressure (symbol: ''p'' or ''P'') is the force applied perpendicular to the surface of an object per unit area over which that force is distributed. Gauge pressure (also spelled ''gage'' pressure)The preferred spelling varies by country and e ...
or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.
Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.
Continuous assays
Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.
Spectrophotometric
In
spectrophotometric
Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry uses photometers, known as spec ...
assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, and these are called colorimetric assays. The
MTT assay
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the t ...
, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.
UV light is often used, since the common coenzymes
NADH
Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an aden ...
and
NADPH
Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NAD ...
absorb UV light in their
reduced forms, but do not in their
oxidized
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
forms. An
oxidoreductase
In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. This group of enzymes usually ut ...
using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.
Direct versus coupled assays
![Coupled assay](https://upload.wikimedia.org/wikipedia/commons/9/9e/Coupled_assay.svg)
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. Here, the product of one reaction is used as the substrate of another, easily detectable reaction. For example, figure 1 shows the coupled assay for the enzyme
hexokinase
A hexokinase is an enzyme that phosphorylates hexoses (six-carbon sugars), forming hexose phosphate. In most organisms, glucose is the most important substrate for hexokinases, and glucose-6-phosphate is the most important product. Hexokina ...
, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using
glucose-6-phosphate dehydrogenase
Glucose-6-phosphate dehydrogenase (G6PD or G6PDH) () is a cytosolic enzyme that catalyzes the chemical reaction
: D-glucose 6-phosphate + NADP+ + H2O 6-phospho-D-glucono-1,5-lactone + NADPH + H+
This enzyme participates in the pentose phosph ...
.
Fluorometric
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
is when a molecule emits light of one
wavelength
In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats.
It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, tro ...
after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light.
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 non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase. Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying
β-galactosidase
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyst, catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosi ...
or 4-methylumbelliferyl-butyrate for assaying ''
Candida rugosa
''Candida'' is a genus of yeasts and is the most common cause of fungal infections worldwide. Many species are harmless commensals or endosymbionts of hosts including humans; however, when mucosal barriers are disrupted or the immune system is ...
''
lipase
Lipase ( ) is a family of enzymes that catalyzes the hydrolysis of fats. Some lipases display broad substrate scope including esters of cholesterol, phospholipids, and of lipid-soluble vitamins and sphingomyelinases; however, these are usually tr ...
.
Calorimetric
Calorimetry
In chemistry and thermodynamics, calorimetry () is the science or act of measuring changes in ''state variables'' of a body for the purpose of deriving the heat transfer associated with changes of its state due, for example, to chemical reacti ...
is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assay in any other way.
Chemiluminescent
Chemiluminescence
Chemiluminescence (also chemoluminescence) is the emission of light (luminescence) as the result of a chemical reaction. There may also be limited emission of heat. Given reactants A and B, with an excited intermediate â—Š,
: + -> lozenge -> ...
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.
The detection of
horseradish peroxidase
The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various or ...
by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in
western blotting
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecti ...
. Another example is the enzyme
luciferase
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''luciferase'', ...
, this is found in fireflies and naturally produces light from its substrate luciferin.
Light scattering
Static light scattering
Static light scattering is a technique in physical chemistry that measures the intensity of the scattered light to obtain the average molecular weight ''Mw'' of a macromolecule like a polymer or a protein in solution. Measurement of the scattering ...
measures the product of weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate.
Hence the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.
Microscale thermophoresis
Microscale thermophoresis
Microscale thermophoresis (MST) is a technology for the Biophysics, biophysical analysis of interactions between biomolecules. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a fun ...
(MST)
measures the size, charge and hydration entropy of molecules/substrates at equilibrium.
The thermophoretic movement of a fluorescently labeled substrate changes significantly as it is modified by an enzyme. This enzymatic activity can be measured with high time resolution in real time.
The material consumption of the all optical MST method is very low, only 5 μl sample volume and 10nM enzyme concentration are needed to measure the enzymatic rate constants for activity and inhibition. MST allows analysts to measure the modification of two different substrates at once (
multiplexing
In telecommunications and computer networking, multiplexing (sometimes contracted to muxing) is a method by which multiple analog or digital signals are combined into one signal over a shared medium. The aim is to share a scarce resource - a ...
) if both substrates are labeled with different fluorophores. Thus substrate competition experiments can be performed.
Discontinuous assays
Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.
Radiometric
Radiometric assays measure the incorporation of
radioactivity into substrates or its release from substrates. The
radioactive isotopes
A radionuclide (radioactive nuclide, radioisotope or radioactive isotope) is a nuclide that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferr ...
most frequently used in these assays are
14C,
32P,
35S and
125I. 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
A scintillation counter is an instrument for detecting and measuring ionizing radiation by using the excitation effect of incident radiation on a scintillating material, and detecting the resultant light pulses.
It consists of a scintillator w ...
.
Chromatographic
Chromatographic assays measure product formation by separating the reaction mixture into its components by
chromatography
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
. This is usually done by
high-performance liquid chromatography
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pa ...
(HPLC), but can also use the simpler technique of
thin layer chromatography
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures.
Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a t ...
. Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher than HPLC instruments (see
High-performance liquid chromatography#Pump pressure).
Factors affecting assays
![Enzyme high pressure b](https://upload.wikimedia.org/wikipedia/commons/7/77/Enzyme_high_pressure_b.jpg)
Several factors effect the assay outcome and a recent review summarizes the various parameters that needs to be monitored to keep an assay up and running.
Salt Concentration
Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak
ionic bonds
Ionic bonding is a type of chemical bonding that involves the electrostatic attraction between oppositely charged ions, or between two atoms with sharply different electronegativities, and is the primary interaction occurring in ionic compounds. ...
of
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
. Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such as the
halophilic
The halophiles, named after the Greek word for "salt-loving", are extremophiles that thrive in high salt concentrations. While most halophiles are classified into the domain Archaea, there are also bacterial halophiles and some eukaryotic species, ...
algae
Algae (; singular alga ) is an informal term for a large and diverse group of photosynthetic eukaryotic organisms. It is a polyphyletic grouping that includes species from multiple distinct clades. Included organisms range from unicellular mic ...
and
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
.
Effects of Temperature
All enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
structure resulting from the breakdown of the weak
ionic and
hydrogen bonding
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a l ...
that stabilize the three-dimensional structure of the enzyme active site. The "optimum" temperature for human enzymes is usually between 35 and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C. Enzymes from
thermophilic
A thermophile is an organism—a type of extremophile—that thrives at relatively high temperatures, between . Many thermophiles are archaea, though they can be bacteria or fungi. Thermophilic eubacteria are suggested to have been among the earl ...
archaea
Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
found in the hot springs are stable up to 100 °C.
However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it would give you low activity at these temperatures.
Effects of pH
Most enzymes are sensitive to
pH and have specific ranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three-dimensional shape of the enzyme by breaking
ionic, and
hydrogen bond
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
s. Most enzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.
Substrate Saturation
Increasing the
substrate concentration increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau.
Level of crowding
Large amounts of
macromolecule
A macromolecule is a very large molecule important to biophysical processes, such as a protein or nucleic acid. It is composed of thousands of covalently bonded atoms. Many macromolecules are polymers of smaller molecules called monomers. The ...
s in a solution will alter the
rates
Rate or rates may refer to:
Finance
* Rates (tax), a type of taxation system in the United Kingdom used to fund local government
* Exchange rate, rate at which one currency will be exchanged for another
Mathematics and science
* Rate (mathema ...
and
equilibrium constant
The equilibrium constant of a chemical reaction is the value of its reaction quotient at chemical equilibrium, a state approached by a dynamic chemical system after sufficient time has elapsed at which its composition has no measurable tendency ...
s of enzyme reactions, through an effect called
macromolecular crowding
The phenomenon of macromolecular crowding alters the properties of molecules in a solution when high concentrations of macromolecules such as proteins are present. Such conditions occur routinely in living cells; for instance, the cytosol of ''Es ...
.
List of enzyme assays
*
MTT assay
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the t ...
*
Fluorescein diacetate hydrolysis
*
''para''-Nitrophenylphosphate
See also
*
Restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
*
DNase footprinting assay
*
Enzyme kinetics
Enzyme kinetics is the study of the rates of enzyme-catalysed chemical reactions. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in th ...
References
External links
*
*
{{Protein methods
Protein methods
assay
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...
Chemical pathology
Clinical pathology
Pathology