Why Isolate Enzyme

❑It is important to study enzymes in a simple system (only with small ions, buffer

molecules, cofactors, etc.) for understanding its structure, kinetics, mechanisms,

regulations, and role in a complex system

❑Also isolating pure enzyme is important to use it for medical and industrial purposes
 Objective of Enzyme Purification
➢ The aim of the purification procedure is to isolate a given enzyme with the maximum

possible yield based on the percentage recovered activity compared with the total

activity in the original extract

➢ In addition the preparation must have the maximum catalytic activity

➢ It should contain no other enzymes or large molecules

➢ If the enzymes to be used as therapeutic purpose, it should be in extremely high

purity and free of pyrogen

 Strategy

1. Source of enzyme

2. Methods of homogenization

3. Methods of separation
 Choice of source
•It is always preferable to select a source enriched in that particular enzyme such as kidney is the
good source of alkaline phosphatase
•It is believed that working with microbial and animal enzymes is easier compared to plant enzymes
since plants are generally rich in phenolics, which on exposure with air get converted into quinones
and quinones bind with enzyme protein and makes it inactive.
•Modern approach with recombinant DNA technology
•Prokaryotes as host organisms (E. coli and Bacillus) preferred for Rapid growth and simple medium
components but lacks post translational modification system and form inclusion bodies
•Yeasts as enzyme source rarely forms inclusion bodies, but grow slowly and have tendency to
hyperglcolysate protein
•‘Fusion Protein’
•Glutathione-S-transferase, maltose binding protein, or His-tag are popularly used
•They greatly enhance the power of purification and sometimes solubility of protein
 Choice of source
❑ Availability
▪ A source with reasonable abundance of the chosen enzyme might not be readily available for
various reasons ( economic, geographical).
▪ In such cases it may be necessary to make some compromise between abundance and availability.
❑ Comparative studies
▪ It may be the case that certain enzyme has been studied in one species or within one tissue in a
species; in such cases it is highly profitable to examine the corresponding enzyme in another
species or another tissue
❑ Subcellular localization
▪ If enzyme occurred in one location within the cell, then the whole tissue can be homogenized or
extracted in purification; sub-cellular fractionation is not necessary
▪ If present in multiple site, sub-cellular fractionation is necessary in order to avoid contamination
from other sources
 Methods of homogenization
❑Mechanical methods

▪ The simplest method is grinding the tissue in a blender with suitable

buffer

▪ Morter and pestle

▪ A gentler technique is to use a potter-Elvejhem homogenizer, a thick

walled test tube through which a tight fitting plunger is passed

▪ Wet grinding by mills or glass balls

❑Non-mechanical methods

Drying, Lysis by osmotic shock, Sonication, detergents, or enzymes,

cycles of freezing and thawing

❑Cooling and protease inhibition are important to recover the enzyme

 Methods of homogenization
❑ If the enzyme is localized in a particular organelle, it is preferable first to isolate the organelle in an
intact form and afterwards conditions may be applied to rupture the organelle. In this process, the
enzyme protein will not get contaminated with the proteins present in other organelles.
❑ If it is a soluble enzyme present in cytoplasm, then generally the complete cell is ruptured without
taking care of getting organelles in intact form.
❑ In case of animal tissues and microbes, many people just use distilled water as isolating
(homogenizing) medium.
❑ However, generally a buffer of a suitable ionic concentration and pH is preferred in order to maintain
the pH and ionic concentration in the medium.
❑ Selection of the pH should be such that isolated enzyme should be in fully active form.
❑ In case of plants, presence of the buffer in the isolating medium is more important since there is
accumulation of acids in plant cell vacuoles and these acids get released in the medium after rupturing.
 Methods of homogenization
❑ The commonly used reducing agents are cysteine, 2-mercaptoethanol (β-mercaptoethanol), sodium
metabisulfite, dithiothreitol, dithioerythritol, reduced glutathione etc. and all are used generally in the
concentration range 10 to 50 mM.
❑ Sometimes, proteases get released from the organelles on rupturing the cells and chew the enzyme protein.
To prevent this a protease inhibitor like phenyl methyl sulfonyl fluoride (PMSF) in 10 to 20 mM
concentration is added in the isolating medium.
❑ A metal chelating agent like ethylene diamine tetra acetate (EDTA) or 2-aminoethyl ether, N, N, N/, N/ tetra
acetate (EGTA) (10 to 50 mM) is added in the isolating medium to prevent the denaturation of the enzyme
protein by heavy metals already present in the tissue/ cell. Sometimes, presence of metal ions like Mg+2,
Mn+2, Zn+2 is essential in the isolating medium for getting the enzyme in the active form.
❑ If one wishes to rupture only the cell wall and cell membrane keeping the cell organelles intact, isotonic
conditions have to be maintained. Generally, 0.25 M mannitol or 0.5 M sucrose is used in the isolating
medium.
❑ Therefore, composition of the isolating medium has to be optimized before starting the actual work.
 Methods of homogenization
❑ Animal cells (organs)
▪ It is easy to homogenize due to the lack of cell wall
▪ Fat and connective tissue must be removed before homogenization
❑ Bacteria and Fungi
▪ Cell wall must be digested by enzymes (Protoplasts can be made by treating
lysozyme or chitinase/3-glucanase)
❑ Plant
▪ Disruption of vacuole can damage enzymes as they are acidic and contain protease
❑ Membrane proteins
▪ Usually detergent (anionic, cationic, or neutral) is added
▪ Detergent must be chosen by considering the choice of purification method
 Methods of Separation
❑ The separation methods can be roughly divided into the following categories:
(i) selective precipitation
(ii) separation based on charge
(iii) separation based on molecular size
(iv) separation based on bio-affinity, and
(v) Separation based on adsorption principles.
❑ Except for the first category, all these methods generally make use of column
chromatography, with column sizes depending on the scale of the sample volumes and
protein concentrations.
❑ The purity of the final enzyme preparation can be tested in several ways. The most common
methods used are sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
analytical gel filtration, and mass spectrometry
 Ammonium sulfate fractionation
❑ This type of fractionation makes use of the fact that individual proteins precipitate at different
concentration ranges of ammonium sulfate
❑ For such an experiment, different amounts of ammonium sulfate (from 0% to 90% saturation) are
added to small samples of cell extract.
❑ Salting out: A large number of water molecules bind with the salt reducing the amount of water
available to interact with the protein molecules.
❑ After addition of the salt, the suspension is stored for few hours in cold condition for complete
precipitation.
❑ After dissolving the ammonium sulfate and removal of the formed protein precipitates by
centrifugation, enzyme activity of the supernatants is measured. Removal of ammonium sulfate
can be accomplished by dialysis, ultrafiltration, or gel filtration
❑ Protein can also be precipitated using organic solvent such as acetone, nonionic polymer such as
PEG
Ammonium sulfate fractionation
Dialysis
 Size-exclusion Chromatography
▪ It is a form of column chromatography in which the stationary phase consists of cross-linked gel
particles
▪ The gel particles are usually in bead form and consist of one of two kinds of polymers
▪ Carbohydrate polymer such as dextran or agarose; trade name Sephadex and Sepharose
▪ Second is based on polyacrylamide
▪ The cross-linked structure of these polymers produces pores in the material

▪ The extent of cross linking can be

controlled to determine the pore size
▪ Large molecules eluted first followed
later by the small ones
▪ Each type of gel has a specific range of
size and exclusion limit
 Affinity Chromatography
▪ It uses specific binding properties of
many proteins
▪ A polymeric material covalently linked
with some compound that bind
specifically with the protein
▪ The other proteins do not bind and
eluted
▪ The bound protein then can be eluted
adding high conc of ligand in soluble
form thus competing for the binding of
protein with stationary phase
▪ The interaction can also be disrupted
with a change in pH or ionic strength
 Ion Exchange Chromatography
▪ IEC is one of the most widely used methods for enzyme purification.
▪ It separates protein molecules according to their differences in charge.
▪ The stationary phase (matrix) in IEC carries charged functional groups fixed by
chemical bonds. The fixed groups are associated with exchangeable counterions.
▪ In anion exchange chromatography, the fixed groups have positive charges and in
cation exchange chromatography, these groups are negatively charged.
▪ As a rule of thumb, proteins bind to an anion exchanger at pH values above their
isoelectric point (pI) and to a cation exchanger at pH values below the pI.
 Ion Exchange Chromatography
▪ Protein IEC usually involves the following steps:
i. Equilibration - The ion exchange resin is equilibrated with a low-salt buffer that allows binding of
the enzyme of interest.
ii. Sample application and adsorption- Protein molecules with a proper charge displace counterions
and bind reversibly to the matrix. The ionic strength of the buffer in which the protein sample is
loaded should be low, as a high concentration of salt usually prevents binding. Any volume of
sample can be applied as long as the total amount of protein does not exceed the binding capacity of
the matrix.
iii. Desorption of bound proteins- A stepwise increase of salt concentration or, in most cases, a gradual
increase of the salt concentration (gradient) of the elution buffer is used.
iv. Cleaning of the column- Proteins and other substances that are bound very strongly to the column
are removed. This is usually done by “cleaning-in-place,”using 2 M NaCl or 0.5 M NaOH solutions,
followed by washing with water/buffer and 20% ethanol for storage.
 Principle
▪ The basic principle involved in ion exchange chromatography is binding of charged proteins on the ion
exchanger by electrostatic attraction (ionic bonds) between charged groups on the proteins and
opposite charges on the exchanger. Conditions like pH are set in such a way that opposite charges be
there between the proteins and ion exchanger. Unbound proteins are removed from the column by
washing with the same medium used for pre-equilibrium. Bound proteins are eluted by passing buffer
of higher ionic strength (using salts like sodium or potassium chloride) or by using buffer of different
pH. It is preferred to make a linear gradient of the salt or pH, instead of step-wise elution. Gradient
elution is considered better since with gradient, there are more chances of removal of unwanted
proteins. Fractions of the effluent are collected and analyzed for the desired enzyme activity.
Purification
 Purification
Total Enzyme activity: This is defined as the total amount of substrate converted to product per
unit time by an enzyme molecule.

𝐶ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (µ𝑚𝑜𝑙𝑒)

𝑇𝑖𝑚𝑒 𝑡𝑎𝑘𝑒𝑛 (𝑚𝑖𝑛)

The unit of enzyme total activity is µmole/min which is also expressed as unit or U

Specific Enzyme activity: The specific activity is the ratio of the enzyme activity to enzyme
concentration.

𝑇𝑜𝑡𝑎𝑙 𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈)

𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 (𝑚𝑔)

Specific activity gives a measurement of enzyme purity in the mixture.

 Purification
Purification fold: Fold purification refers to the number of times a protein preparation is
enriched for the protein being purified. If a protein has a concentration of 1mM before
purification and a concentration of 10mM after purification the fold purification is 10 fold.

𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑎𝑓𝑡𝑒𝑟 𝑝𝑢𝑟𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛

𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝑐𝑟𝑢𝑑𝑒 𝑝𝑟𝑒𝑝𝑎𝑟𝑎𝑡𝑖𝑜𝑛

Yield or Recovery: The yield is a measure of how much enzyme activity has retained in the
sample that you have purified. It is equal to the ratio of the enzyme activity of that sample to the
enzyme activity of the original sample multiplied by 100%.

𝑇𝑜𝑡𝑎𝑙 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑖𝑛 𝑝𝑢𝑟𝑖𝑓𝑖𝑒𝑑 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛

x100%
𝑇𝑜𝑡𝑎𝑙 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑖𝑛 𝑡ℎ𝑒 𝑐𝑟𝑢𝑑𝑒 𝑝𝑟𝑒𝑝𝑎𝑟𝑎𝑡𝑖𝑜𝑛
Purity Check
 Enzyme Assay
❑ Enzyme assays are performed to serve two different purposes:
(i) to identify a special enzyme, to prove its presence or absence in a distinct specimen,
like an organism or a tissue and
(ii) to determine the amount of the enzyme in the sample
❑ First, the qualitative approach, a clear positive or negative result is sufficient, the
second, the quantitative approach must deliver data as exact as possible.
❑ Enzymes display their highest activity at their respective optimum conditions,
deviations from the optimum cause a reduction of the activity, depending on the
degree of the deviation.
 General Consideration
❑ The predominant rule is the clear and easy mode of observation of the enzyme
reaction.
❑ In assay, a substrate is converted to product. A procedure must be found to identify
the product formed during the assay.
❑ A simple but important condition is that substrate and product must differ in the
observed feature. The product may be very well detectable by a distinct method, but
if the substrate shows a similar signal with equal intensity, no turnover can be
observed at all.
❑ In such cases the signal to noise ratio must be analysed
❑ As a rule the intensity of the signal displayed by the reaction must exceed the noise at
least by a factor of two.
General Consideration
 Methods
❑ Due to the relative easy handling and the low susceptibility against disturbances
photometric assays are applied as far as possible
❑ If an enzyme reaction cannot be observed photometrically, other optical methods may
be used. Fluorimetry is more sensitive than absorbance measurements (about hundred
fold), but only a few enzymatic substrates or products emit fluorescence
❑ Spectrofluorimeters are more complicated to handle and there exist more sources for
errors, therefore fluorimetric assays are unusual, and a deeper experience is needed
❑ Besides optical methods, electrochemical methods are in use, especially pH
determinations for reactions proceeding with pH changes,
 Stopped assay
❑To measure the activity of an enzyme one must measure how much product is formed
over a given time or, in some cases, how much substrate has been used up, which
should be the same thing.
❑Stopped assays involve stopping the reaction after a fixed time, then measuring how
much product has been formed
❑Methods for stopping the reaction include those which denature the enzyme, such as
strong acid, alkali or detergent; heat; or treatments with irreversible inhibitors such as
heavy metal ions.
❑In some cases the enzyme can be stopped by addition of a complexing agent such as
ethylenediaminetetraacetic acid (EDTA), which removes metal ions essential for
activity; even chilling on ice may be sufficient.
 Continuous assay
❑ Continuous assays are much more convenient in that the result is seen immediately,
and any deviation the initial rate shows from linearity can be observed.
❑ On the other hand not all enzymes have an assay method that can be observed
continuously.
❑ The simplest continuous assay is one in which the action of the enzyme itself can be
followed by changes in absorbance (e.g. NADPH at 340nm with dehydrogenases),
fluorescence, viscosity, pH, or one of several other possible physical parameters