General techniques for identification and characterization of protein and Nucleic acid.pptx
1. General techniques used for
identification and characterization of
proteins and nucleic acids
2. Measurement of Protein
Biochemical research often requires quantitative measurement of protein in solutions.
Several techniques have been developed; however, most have limitations
Amino acid content varies from protein to protein, no single assay will be suitable.
Here, we discuss five assays, in most methods chemical reagents are added to protein
solutions to develop a color whose intensity is measured in a spectrophotometer.
A “standard protein” of known concentration is also treated with same reagents & a
calibration curve is constructed.
The other assay relies on direct spectrophotometric measurement.
The various methods are compared in Table 3.3.
3.
4. Biuret Assays
Substances containing two or more peptide bonds react with biuret reagent, alkaline
copper sulfate, a purple complex is formed.
The colored product is the result of coordination of peptide nitrogen atoms with Cu+2.
The amount of product formed depends on the concentration of protein.
Calibration curve must be prepared by using a standard of protein to be assayed.
A relative standard must be selected that provides a similar color yield.
An aqueous solution of bovine serum albumin (BSA) is a commonly used standard.
Measurements of absorbance at 540 nm (A540) are made against a blank.
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The data are plotted versus protein concentration (mg/mL) or amount of protein (mg).
Unknown protein samples are treated with biuret reagent & the (A540) is measured after
color development.
The protein concentration is determined from the standard curve.
The biuret assay has several advantages, including speed, similar color development
with different proteins, and few interfering substances.
Its primary disadvantage is its lack of sensitivity.
6. Lowry Protein Assay
Principle behind color development is identical to that of biuret assay except that a second
reagent (Folin-Ciocalteu) is added to increase the amount of color development.
Two reactions account for intense blue color that develops through
the coordination of peptide bonds with alkaline copper (biuret reaction),
reduction of Folin-Ciocalteu reagent by tyrosine & tryptophan residues in protein.
The procedure is similar to that of biuret assay.
Obvious advantage of Lowry assay is its sensitivity however, more time is required.
Since proteins have varying contents of tyrosine and tryptophan, the amount of color
development changes with different proteins, including the bovine serum albumin standard.
For this, the Lowry protein assay should be used only for measuring changes in protein
concentration, not absolute values of protein concentration.
7. Bradford Assay
This assay is based on protein binding of a dye, provides numerous advantages over
other methods.
Binding of Coomassie Brilliant Blue dye (Figure 3.5) to protein in acidic solution causes
a shift in wavelengh of maximum absorption (λmax) of dye from 465 nm to 595 nm.
The absorption at 595 nm is directly related to the concentration of protein.
The dye reagent interacts primarily with positively-charged A. A in proteins.
In addition, there are probably weaker interactions between the dye molecules &
hydrophobic, aromatic amino acid residues.
Proteins that have more than an average number of these interacting residues show
higher sensitivity and thus higher absorbance values at 595 nm.
8. Calibration curve is prepared by plotting µg of standard protein versus absorbance at
595 nm, used to estimate amount of protein in unknown samples (Figure 3.6).
Recommended standard proteins include bovine gamma globulin (IgG), lysozyme, &
ovalbumin as closer to average or typical number of A.A. residues that bind the dye.
Although BSA is often recommended in the literature, it is not a good choice.
The 595 nm absorbance of BSA is about 2.1 times that of IgG (Figure 3.6).
This assay requires only a single reagent, i.e. Coomassie Brilliant Blue G-250.
After addition of dye solution to a protein sample, color development is complete in 2
minutes & the color remains stable for up to 1 hour.
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9. Sensitivity of Bradford assay rivals & may surpass that of the Lowry assay.
With a microassay procedure, the Bradford assay can be used to determine proteins in
the range of 1 to 20 µg.
Bradford assay shows significant variation with different proteins, but this also occurs
with Lowry assay & can be avoided by use of the proper protein standard.
Bradford method not only is rapid, but also has very few interferences by non-protein
components.
The only known interfering substances are the detergents Triton X-100 & sodium
dodecyl sulfate.
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10.
11.
12. BCA Assay
The BCA protein assay is based on chemical principles similar to those of biuret &
Lowry assays.
Protein to be analyzed is reacted with Cu+2 & bicinchoninic acid (BCA).
The Cu+ is chelated by BCA, converts the apple-green color of free BCA to purple color
of copper- BCA complex.
Samples of unknown protein & relative standard are treated with the reagent, the
absorbance is read in a spectrophotometer at 562 nm
Concentrations are determined from a standard calibration curve.
This assay has the same sensitivity level as the Lowry & Bradford assays.
Main advantages are its simplicity & usefulness in the presence of 1% detergents such
as Triton or sodium dodecyl sulfate.
13. Spectrophotometric Assay
Most proteins have relatively intense ultraviolet light absorption centered at 280 nm.
Absorbance taken at A280 gives value of a protein that is proportional to protein conc.
In practice, the procedure is simple and rapid.
A protein solution is transferred to a quartz cuvette & measure its absorbance at A280
against a reference cuvette containing the protein solvent only.
For average, a conc. of 1 mg/mL will yield an absorbance at 280 nm of about 1.
If the extinction coefficient is known for protein, its exact concentration may be
calculated from absorbance at 280 nm.
Cellular extracts contain many other compounds that absorb in the vicinity of 280 nm.
Nucleic acids, which can be common contaminants in a protein extract, absorb strongly
at 280 nm (λmax = 260).
14. Early researchers developed a method to correct for this interference by nucleic acids.
Mixtures of pure protein & pure nucleic acid were prepared, and the ratio was
experimentally determined.
The following empirical equation may be used for protein solutions containing up to 20%
nucleic acids. “Protein conc. (mg/ml) = 1.55A280 - 0.76A260”
Although, this method is fast, relatively sensitive, & requires only a small sample size, it
is still only an estimate of protein conc. unless its extinction coefficient is known.
It has certain advantages over colorimetric assays i.e. most buffers & ammonium sulfate
do not interfere & the procedure is nondestructive.
It is particularly suited for rapid & continuous measurement of protein elution from a
chromatography column, where only protein concentration changes are required.
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15. Measurement of Nucleic Acid (N.A)
Spectrophotometric Assay
Solutions of N.A strongly absorb ultraviolet light with a of λmax 260 nm.
Intense absorption is due to the presence of aromatic rings in purine & pyrimidine bases.
Conc. of N.A in solution can be calculated if one the knows value of A260 of solution.
Solution of double stranded DNA at conc. of 50 mg>mL in a 1-cm quartz cuvette will give
a reading of about 1.0.
A solution of single-stranded DNA or RNA that has an A260 reading of 1.0 in a cuvette
with a 1-cm path length has a concentration of about 40 mg>mL
It is simple, rapid, nondestructive, and very sensitive assay; however, it should be noted
that it measures both DNA and RNA.
16. The minimum conc. of N.A that can be accurately determined is 2.5–5.0 mg>mL.
Measuring the ultraviolet absorption of DNA & RNA solutions can also provide a useful
structural probe & an indication of purity.
DNA structural changes such as helix unwinding may be detected by measuring
absorbance changes of DNA solutions at 260 nm.
Ratio of A260/A280 is often used as a relative measure of purity of N.A solutions.
Pure DNA with little or no protein present has an absorbance ratio of about 1.8.
Pure RNA has a ratio about 2.
Absorbance ratios less then these indicate contamination of solution by protein material.
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17. Other Assays for N.A
Spectrophotometric assay does not have a sufficient level of sensitivity for many
applications.
Other assays, depend on enhanced fluorescence of dyes when bound to N.A.
Features of fluorescence assays are compared to spectrophotometric assay in Table 3.4
The fluorescence dye bisbenzimidazole, when bound to DNA, emits light at 458 nm that
is about five times greater in intensity than emission by free, unbound dye.
Increase in fluorescence is linear with increasing amounts of DNA in conc. range of 0.01
to 5 µg/ ml.
18. Advantage of this assay is that it can quantify DNA in cell suspensions & crude
homogenate
It does not respond to RNA or other biomolecules in extracts.
The is fast & requires only a fluorimeter.
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21. Dialysis
Oldest procedures applied to the purification & characterization of biomolecules is
dialysis
This involves sealing an aqueous solution containing both macromolecules & small
molecules in a porous membrane.
Sealed membrane is placed in a large container of low-ionic-strength buffer.
The membrane pores are too small to allow diffusion of macromolecules of M.W greater
than about 10,000.
Smaller molecules diffuse freely through the openings (Figure 3.7).
Passage of smaller molecules continues until their conc. inside the dialysis tubing &
outside in large volume of buffer are equal.
22. Thus, the conc. of small molecules inside the membrane is reduced.
Equilibrium is volume dependent & reached after 4 to 6 hours.
If dialysate is replaced with fresh buffer after equilibrium, the conc. of small molecules
inside membrane will be further reduced by continued dialysis.
Dialysis is commonly used to remove salts & other small molecules from solutions of
macromolecules.
During separation & purification of biomolecules, small molecules are added to
selectively precipitate or dissolve the desired molecule.
Dialysis is simple, inexpensive, & effective method for removing small molecules.
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23. Also useful for removing small ions & molecules that are weakly bound to biomolecules.
Protein cofactors like NAD, FAD, & metal ions can often be dissociated by dialysis.
Removal of metal ions is facilitated by addition of a chelating agent to dialysate.
Dialysis of small-volume samples is often tedious, inconvenient, & inefficient.
Commercial vials for dialysis are now available for sample sizes from 0.05 to 3.0 mL,
with molecular weight cutoffs ranging from 3500 to 14,000 daltons.
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24.
25. Ultrafiltration
Ultrafiltration involves separation of molecular species on the basis of size, shape,
and/or charge.
The solution to be separated is forced through a membrane by an external force.
Membranes may be chosen for optimum flow rate, molecular specificity, & molecular
weight cutoff.
Membrane filtration mostly applied for
desalting buffers or other solutions
clarification of turbid solutions by removal of micron- or submicron-sized particles.
Ultrafiltration membranes have M.W cutoffs in the range of 100 to 1,000,000.
26. They are usually composed of two layers
a thin surface that is semipermeable membrane made from a variety of materials
including cellulose acetate, nylon, & polyvinylidene
a thicker, inert, support base (Figure 3.8)
These filters function by retaining particles on surfaces, not within the base matrix.
Membrane filters of these materials can be manufactured with a predetermined &
accurately controlled pore size.
Filters are available with a mean pore size ranging from 0.025 to 15µm.
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27. These filters require suction, pressure, or centrifugal force for liquid flow.
Typical flow rate for commonly used 0.45–µm membrane is 57mL/min-1/cm-2 at 10 psi.
Ultrafiltration devices are available for macroseparations (up to 50 L) or for
microseparations (milli- to microliters).
For solutions larger than a few milliliters, gas-pressurized cells or suction-filter devices
are used.
For concentration & purification of samples in the milli- to microliter range, disposable
filters are available.
These devices, often called microconcentrators, offer user simplicity, time saving, & high
recovery.
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28. The sample is placed in a reservoir above the membrane & centrifuged.
The time & centrifugal force required depend on the membrane.
Figure 3.9 outlines the use of a centrifuge microfilter.
The nomenclature implies that separations are result of physical trapping of particles &
molecules by filter.
With polycarbonate & fiberglass filters, separations are made primarily on the basis of
physical size.
Other filters trap particles that cannot pass through the pores, but also retain
macromolecules by adsorption.
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29. In particular, these materials have protein & nucleic acid-binding properties.
Each type of membrane displays a different affinity for various molecules.
For protein, the relative binding affinity is polyvinylidene fluoride ˃ cellulose nitrate ˃
cellulose acetate.
Some applications of ultrafiltration are here
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30.
31. Clarification of Solutions
Due to low solubility & denaturation, solutions of biomolecules or cellular extracts are
often turbid.
This is a particular disadvantage if spectrophotometric analysis is desired.
Transmittance of turbid solutions can be greatly increased by passage through a
membrane filter system.
This simple technique may also be applied to the sterilization of non-autoclavable
materials such as protein & nucleic acid solutions or heat-labile reagents.
Bacterial contamination can be removed from these solutions by passing them through
filter systems that have been sterilized by autoclaving.
32. Collection of Precipitates for Analysis
Collection of small amounts of very fine precipitates is the basis for many chemical &
biochemical analytical procedures.
Membrane filtration is an ideal method for sample collection.
This is of great advantage in the collection of radioactive precipitates.
Cellulose nitrate and fiberglass filters are often used to collect radioactive samples
because they can be analyzed by direct suspension in an appropriate scintillation
cocktail.
33. Harvesting Bacterial Cells
Collection of bacterial cells from nutrient broths is typically done by batch centrifugation.
This time-consuming operation can be replaced by membrane filtration.
Filtration is faster than centrifugation, & allows extensive cell washing.
34. Concentration of Biomolecule
For concentration of biomolecules, ultrafiltration pressure cells are most effective (Fig
3.10).
A membrane filter is placed in bottom & the solution is poured into the cell.
High pressure, exerted by compressed nitrogen, forces the flow of small molecules,
including solvent, through the filter.
Larger molecules that cannot pass through pores are concentrated in sample chamber.
This method of concentration is rapid & gentle and can be performed at cold
temperatures to ensure minimal inactivation of the molecules.
One major disadvantage is clogging of pores, which reduces the flow rate through the
filter; this is lessened by constant but gentle stirring of solution.
35.
36. Lyophilization
Lyophilization is used for concentration of biological solutions.
It is a drying technique that uses to change a solvent in frozen state directly to vapor
state under vacuum.
The product after lyophilization is a fluffy matrix.
This is the effective methods for drying or concentrating heat-sensitive materials.
In practice, a biological solution to be concentrated is “shell-frozen” on the walls of a
round-bottom or freeze-drying flask.
Freezing of solution is accomplished by placing the flask in a dry ice–acetone bath &
slowly rotating it as it is held at an angle of 45 degree.
37. Aqueous solution freezes in layers on the wall of the flask.
This provides a large surface area for evaporation of water.
Flask is then connected to lyophilizer, which consists of a refrigeration unit & vacuum
pump (Figure 3.11).
Combined unit maintains the sample at -40°C for stability of biological materials &
applies a vacuum of approximately 5 to 25 mm Hg on sample.
Ice formed from aqueous solution sublimes & is pumped from the sample vial.
In fact, all materials that are volatile under these conditions (-40°C, 5 to 25 mm Hg) will
be removed, and nonvolatile materials will be concentrated into a light, sometimes fluffy
precipitate.
Most freeze-dried biological materials are stable for long periods & some remain viable
for many years.
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39. Only aqueous solutions should be lyophilized.
Organic solvents lower the melting point of aqueous solutions & increase the chances
that the sample will melt & become denatured during freeze-drying.
There is also the possibility that organic vapors will pass through the cold trap into the
vacuum pump, where they may cause damage.
A new & increasingly popular technique for sample concentration & drying is centrifugal
vacuum concentration.
Used to dry a wide variety of biological samples.
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40. The process starts with a sample dissolved in a solvent.
Solvent is evaporated under vacuum in a centrifuge, thus producing a pellet in the
bottom of container.
The most widely used instrument is the SpeedVac, available through Savant (Figure
3.12).
This method is better than freeze-drying because it is faster, it does not require a
prefreezing step
It provides 100% sample recovery
May be used with solvents other than water.
The SpeedVac is usually used for relatively small volumes—1–2-mL samples.
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41.
42. Centrifugation
Centrifugation is carried out by spinning a biological sample at a high rate of speed, thus
subjecting it to an intense force.
Most centrifuge techniques fit into one of two categories—preparative or analytical
centrifugation.
A preparative procedure is one that can be applied to the separation or purification of
biological samples by sedimentation.
Analytical procedures are used to measure physical characteristics of biological
samples.
43. Instrumentation for Centrifugation
Basic centrifuge consists of two components, an electric motor with drive shaft to spin
the sample & a rotor to hold tubes or other containers of sample.
A wide variety of centrifuges is available like the low-speed or clinical centrifuge; the
high speed centrifuge, including the “microfuge”; & the ultracentrifuge.
Major characteristics & applications of each type are compared in Table 4.1.
Here we discuss ultracentrifuge in detail.
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45. Ultracentrifuge
Most sophisticated centrifuges are the ultracentrifuges.
Spin chamber must be refrigerated & placed under a high vacuum to reduce friction.
Sample in a cell or tube is placed in rotor, which is then driven by an electric motor.
Although it is rare, metal rotors sometimes break into fragments when placed under high
stress.
Rotor chamber on all ultracentrifuges is covered with a protective steel armor plate.
The drive shaft of ultracentrifuge is constructed of a flexible material to accommodate
any “wobble” of rotor due to imbalance of samples.
It is important to counterbalance samples before inserting them in the rotor.
46. The centrifuge i.e. low, medium, & high speed are of value only for preparative work.
Ultracentrifuges can be used both for preparative work & analytical measurements.
preparative models; primarily used for separation & purification of samples
analytical models; designed for performing physical measurements on the sample
during sedimentation.
Two of most versatile models are Beckman Optima MAX & TLX microprocessor-
controlled tabletop ultracentrifuges (Figure 4.10).
With a typical fixed-angle rotor, which holds six 0.2- to 2.2-mL samples, can generate
150,000 rpm & an RCF of up to 1,500,000.
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47. Analytical ultracentrifuges have same basic design as preparative models, except that
they are equipped with optical systems to monitor directly the sedimentation of sample
during centrifugation.
For analysis, a sample of nucleic acid or protein (0.1 to 1.0 mL) is sealed in a special
analytical cell & rotated.
Light is directed through the sample parallel to the axis of rotation, & measurements of
absorbance by sample molecules are made.
If sample molecules have no significant absorption bands in the wavelength range, then
optical systems that measure changes in the refractive index may be used.
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48. Optical systems aided by computers are capable of relating absorbance changes or
index of refraction changes to the rate of movement of particles in sample.
The optical system actually detects & measures the front edge or moving boundary of
the sedimenting molecules.
These measurements can lead to an analysis of concentration distributions within the
centrifuge cell.
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50.
51. Recommended book
Biochemistry Laboratory: Modern Theory and
Techniques
Rodney Boyer (Hope College)
Second Edition
Chapter 3 and 4:General laboratory procedures and
Centrifugation techniques in biochemistry