Download Charge:-Protein

25. Methods of protein study.
 Physic- chemical properties of proteins useful in their separation:- salting-out,
denaturation, charge, isoelectric point, utilization of specificity of proteases.
 Principles of protein separation: - Electrophoresis, chromatography, and
sequencing.
Salting-out:Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations. The salt concentration needed for
the protein to precipitate out of the solution differs from protein to protein. This
process is also used to concentrate dilute solutions of proteins.
Hydrophobic and hydrophilic aminoacids are present in protein molecules.
After protein folding in aqueous solution, hydrophobic amino acids usually form
protected hydrophobic areas while hydrophilic amino acid interact with the molecules
of solvation and allow proteins to form hydrogen bonds with the surrounding water
molecules. Protein can be dissolved in water, if the protein surface is hydrophilic.
When the salt concentration is increased, some of the water molecules are
attracted by the salt ions, which decreases the number of water molecules available to
interact with the charged part of the protein. As a result of the increased demand for
solvent molecules, the protein-protein interactions are stronger than the solvent-solute
interactions; the protein molecules coagulate by forming hydrophobic interactions with
each other. This process is known as salting out.
Solvation:-A sodium
ion solvated by water
molecules
Denaturation:Denaturation of proteins involves the disruption and possible destruction of
both the secondary and tertiary structures. Since denaturation reactions are not strong
enough to break the peptide bonds, the primary structure (sequence of amino acids)
remains the same after a denaturation process. Denaturation disrupts the normal alphahelix and beta sheets in a protein and uncoils it into a random shape.
Denaturation occurs because the bonding interactions responsible for the secondary
structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary
structure there are four types of bonding interactions between "side chains" including:
hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic
interactions. Which may be disrupted. Therefore, a variety of reagents and conditions
can cause denaturation. The most common observation in the denaturation process is
the precipitation or coagulation of the protein.
Charge:-Protein
The charge on the protein affects its behavior in ion exchange chromatography. Proteins
contain many ionizable groups on the side chains of their amino acids as well as their
amino - and carboxyl - termini. These include basic groups on the side chains of lysine,
arginine and histidine and acidic groups on the side chains or glutamate, aspartate,
cysteine and tyrosine. The pH of the solution, the pK of the side chain and the side
chain’s environment influence the charge on each side chain. The relationship between
pH, pK and charge for individual amino acids can be described by the HendersonHasselbalch equation
In general terms, as the pH of a solution increases, deprotonation of the acidic and basic
groups on proteins occur, so that carboxyl groups are converted to carboxylate anions
(R-COOH to R-COO-) and ammonium groups are converted to amino groups (R-NH3+ to
R-NH2). In proteins the isoelectric point (pI) is defined as the pH at which a protein has
no net charge. When the pH > pI, a protein has a net negative charge and when the pH <
pI, a protein has a net positive charge. The pI varies for different proteins.
The charge on proteins arises from some of the amino acid side chains, as well as the
carboxy- and amino-termini, some prosthetic groups and bound ions. This application is
designed to calculate charge based only on the side chains and carboxy - and aminotermini.
The charge on amino acid side chains depends on the pH of the solution and the pKA of
the side chains. It is also affected by the localized environment around a side chain. We
assume the following pKA values for ionizable groups on the protein and that the side
chains will have these pKA values regardless of their environment within the protein. We
also assume that the separation is based on the total charge on the protein, not the
mass-to-charge ratios. Therefore, a protein with a charge of +15 will bind more tightly to
a cation exchange stationary phase than a protein with a charge of +10, regardless of
size.
Group
pKA
Acids
Carboxy-terminus 3.1
Aspartate
4.4
Glutamate
4.4
Cysteine
8.5
Tyrosine
10.0
Bases
Amino-terminus 8.0
Lysine
10.0
Arginine
12.0
Histidine
6.5
In short:-when the pH is less than the pKA of a group, the
protonated form of the group predominates. This leaves the
acidic side chains with a charge approaching 0 and the basic
side chains with a charge approaching a limiting value of +1.
Conversely, when the pH is greater than the pKA of a group,
the deprotonated form predominates, giving acidic side chains
a charge approaching -1 and basic side chains a charge
approaching 0.
The charge on the protein is the sum of the charges on the
individual amino acid side chains. However, the charge on
individual amino acid side chains can vary when they are near
a group of non-polar or highly charged side chains.
Isoelectric point:The isoelectric point (IEP), is the pH at which a particular molecule or surface carries no
net electrical charge.
Amphoteric molecules which are also known as zwitterions contain both +ve and -ve
charges depending on the functional groups present in the molecule. The net charge on
the molecule is affected by pH of their surrounding environment and can become more
positively or negatively charged due to the loss or gain of protons (H+). The isoeletric
point is pH value at which the molecule carries no electrical charge or the negative and
positive charges are equal.
Surfaces naturally charge to form a double layer. In the common case when the surface
charge-determining ions are H+/OH-, the net surface charge is affected by the pH of the
liquid in which the solid is submerged. Again, the isoeletric point is the pH value of the
solution at which the surfaces carries no net charge.
The isoeletric point value can affect the solubility of a molecule at a given pH. Such
molecules have minimum solubility in water or salt solutions at the pH which
corresponds to their isoeletric point and often precipitate out of solution. Biological
amphoteric molecules such as proteins contain both acidic and basic functional groups.
Amino acids which make up proteins may be positive, negative, neutral or polar in
nature, and together give a protein its overall charge. At a pH below their isoelectric
point, proteins carry a net positive charge; above their pI they carry a net negative
charge. Proteins can thus be separated according to their isoelectric point (overall
charge) on a polyacrylamide gel using a technique called isoelectric focusing, which uses
a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel
polyacrylamide gel electrophoresis.
(also read from practical notes”eletrophresis page 6”)
 Principles of protein separation
Chromatography:- It is a separation method based on the different migration of solutes
through a system of two diverse phases, one of which is mobile and the other
stationary.
Chromatographic methods can be classified according to:
A. Mobile phase arrangement
• Liquid chromatography (LC) - mobile phase is a liquid
• Gas chromatography (GC) - mobile phase is a gas.
B. Stationary phase arrangement
• Column chromatography – stationary phase is placed in a column
• Planar techniques:
Paper chromatography (PC) – stationary phase is a special paper, either as such or
modified with other compounds.
Thin-layer chromatography (TLC) – stationary phase is spread on a solid flat
support (e.g., glass plate or aluminum foil)
C. The process, which prevails in separation(usually several physical and chemical
processes take place in separation but one of them
prevails)
• Partition chromatography – separation is based on different solubility of sample
components in a stationary phase (a liquid) and in a mobile phase (a liquid or a gas).
• Adsorption chromatography – separation is based on different abilities of components
to adsorb on the surface of stationary phase (a solid).
• Ion-exchange chromatography – separation is based on exchange of the ionic sample
with the ionic group of the stationary phase and is governed by electrostatic interaction.
• Size exclusion chromatography (gel chromatography) – components are separated
according to the size and shape of their molecules as well as the size and shape of the
pores of the stationary phase (size-exclusion effect). The large molecules elute at the
beginning, and the small molecules at the end.
• Affinity chromatography – separation is based on molecular recognition. Only those
components, which are complementary to stationary phase, are adsorbed by their
affinity.Affinity interactions are very strong.
(In detail - check practical lab note topic “chromatography”)
Electrophoresis:- (read practical notes topic “Electrophoresis”)
Sequencing:Its major aim is to determine the primary sequence or primary structure of an
unbranched biopolymer. Proteins and peptides, and DNA & RNA all are the
examples of biopolymer which is a class of polymers produced by living organisms, in
which monomeric units are sugars, amino acids and nucleotides.
Pyrosequencing:Pyrosequencing, which was originally developed by Mostafa Ronaghi, has been
commercialized by Biotage (for low throughput sequencing) and 454 Life Sciences (for
high-throughput sequencing). The latter platform sequences roughly 100 megabases in
a 7-hour run with a single machine. In the array-based method, single-stranded DNA is
annealed to beads and amplified via EmPCR. These DNA-bound beads are then placed
into wells on a fiber-optic chip along with enzymes which produce light in the presence
of ATP. When free nucleotides are washed over this chip, light is produced as ATP is
generated when nucleotides join with their complementary base pairs. Addition of one
(or more) nucleotide(s) results in a reaction that generates a light signal that is recorded
by the CCD camera in the instrument. The signal strength is proportional to the number
of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide
flow.
Sanger sequencing (chain termination method)
Developer -Frederick Sanger
The
key
principle
of
the
Sanger
method
was
the
of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.
use
In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific
site on the template DNA by using a short oligonucleotide 'primer' complementary to
the template at that region. The oligonucleotide primer is extended using a DNA
polymerase, an enzyme that replicates DNA. Included with the primer and DNA
polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low
concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide).
Limited incorporation of the chain terminating nucleotide by the DNA polymerase
results in a series of related DNA fragments that are terminated only at positions where
that particular nucleotide is used. The fragments are then size-separated by
electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass
tube (capillary) filled with a viscous polymer.
An alternative to the labelling of the primer is to label the terminators instead,
commonly called 'dye terminator sequencing'. The major advantage of this approach is
the complete sequencing set can be performed in a single reaction, rather than the four
needed with the labeled-primer approach. This is accomplished by labelling each of the
dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces
at a different wavelength. This method is easier and quicker than the dye primer
approach, but may produce more uneven data peaks (different heights), due to a
template dependent difference in the incorporation of the large dye chain-terminators.
This problem has been significantly reduced with the introduction of new enzymes and
dyes that minimize incorporation variability.
This method is now used for the vast majority of sequencing reactions as it is both
simpler and cheaper. The major reason for this is that the primers do not have to be
separately labelled (which can be a significant expense for a single-use custom primer),
although this is less of a concern with frequently used 'universal' primers.
Dye-terminator sequencing:Dye-terminator sequencing utilizes labeling of the chain terminator ddNTPs, which
permits sequencing in a single reaction, rather than four reactions as in the labeledprimer method. In dye-terminator sequencing, each of the four dideoxynucleotide
chain terminators is labeled with fluorescent dyes, each of which with
different wavelengths of fluorescence and emission. Owing to its greater expediency
and speed, dye-terminator sequencing is now the mainstay in automated
sequencing. Its limitations include dye effects due to differences in the incorporation
of the dye-labeled chain terminators into the DNA fragment, resulting in unequal
peak
heights
and
shapes
in
the
electronic
DNA
sequence
trace chromatogram after capillary electrophoresis. This problem has been
addressed with the use of modified DNA polymerase enzyme systems and dyes that
minimize incorporation variability, as well as methods for eliminating "dye blobs".
The dye-terminator sequencing method, along with automated high-throughput
DNA sequence analyzers, is now being used for the vast majority of sequencing
projects.
Maxam-Gilbert sequencing:The method requires radioactive labeling at one end and purification of the DNA
fragment to be sequenced. Chemical treatment generates breaks at a small proportion
of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, and C+
T). Thus a series of labeled fragments is generated, from the radio labeled end to the
first 'cut' site in each molecule. The fragments in the four reactions are arranged side by
side in gel electrophoresis for size separation. To visualize the fragments, the gel is
exposed to X-ray film for autoradiography, yielding a series of dark bands each
corresponding to a radio labeled DNA fragment, from which the sequence may be
inferred.
Also sometimes known as 'chemical sequencing', this method originated in the study of
DNA-protein interactions (foot printing), nucleic acid structure and epigenetic
modifications to DNA, and within these it still has important applications.