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Biological Macromolecules
Nucleic Acids
Like many biological molecules nucleic acids are polymers, long molecules formed of repeating units. With
nucleic acids, the repeating unit is the nucleotide. A nucleotide consists of a five carbon sugar, a nitrogen
containing base and a phosphate group. The two primary kinds of nucleic acids, deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA), possess slightly different sugars in their respective nucleotides and a
different set of four bases which may be contained by their nucleotides.
DNA Nucleotide
RNA nucleotide
The structure of a section of an RNA
molecule.
Note the presence of a hydroxyl group on the 2' carbon of the
sugar moety.
Adenine DNA and RNA
Guanine DNA and RNA
Thymine DNA and RNA
Cytosine DNA and RNA
Uracil RNA only
Of great importance to electrophoresis is the ionization of the phosphate groups, giving nucleic acids a large
net negative charge. Because each nucleotide is ionized, the charge to mass ratio of two different nucleic
acid molecules will very closely agree.
The phosphate groups of each nucleotide carry a negative formal charge. Because each nucleotide carries the
same charge, the charge to mass ratio of different nucleic acid molecules are nearly identical. Electric force
causes negatively charged nucleic acid molecules to migrate toward the positive pole.
DNA and RNA each contain four possible nucleotides corresponding to the set of four possible bases
(adenine, guanine, thymine and cytosine for DNA; adenine, guanine, uracil, and cytosine for RNA). Each
base exhibits a particular affinity for one of the other three bases, based on hydrogen bonding symmetries.
The nitrogen base adenine "base pairs" with thymine (or uracil in RNA). Guanine "base pairs" with cytosine.
Because of base pairing, DNA or RNA can exist as single stranded or double stranded variants. The double
stranded form consists of two complementary strands joined by base pairing.
The base pairing of two complementary strands allows nucleic acid molecules to assume a double stranded
form.
Base pairing can also occur in single stranded DNA or RNA. A section containing one sequence of
nucleotides will often loop back and base pair with a complementary section on the same chain. This will
affect the 3 dimensional structure of the molecule, with implications for electrophoretic separations. In
general, long strands of DNA or RNA will be found in a base-paired conformation, either double stranded or
single stranded with internal pairing. Unpaired, or "denatured" nucleic acids are only found in solution under
special conditions which destabilize the base pairs.
Base pairing is not limited to double stranded variants, but can also occur within the same molecule. The
resulting conformations can lead to electrophoresis results that are difficult to interpret.
Electrophoresis of double stranded DNA or RNA is referred to as native gel electrophoresis. Electrophoresis
of single stranded DNA or RNA occurs under denaturing conditions. Formamide and urea are the two most
common agents which accomplish chemical denaturation. These substances act to disrupt the hydrogen
bonding between the nitrogen bases, thereby removing the effects of base pairing. Usually some
combination of formamide, urea, and heat is employed over the process of denaturing electrophoresis from
sample preparation to running the gel. The purposes of denaturing conditions are to ensure single stranded
molecules and to prevent conformational changes due to base pairing between different sections of the same
DNA or RNA molecule. Denaturing electrophoresis conditions allow for a consistent relationship between
molecular size and mobility through the gel.
Formamide and urea accomplish the denaturation of DNA or RNA by forming new hydrogen bonds with the
bases of the nucleic acid molecules,disrupting the hydrogen bonds that lead to base pairing.
Proteins
Like nucleic acids, proteins are polymers. While with nucleic acids the repeating unit is the nucleotide, with
proteins, the analogous repeating unit is the amino acid. Amino acids consist of a central carbon which
carries an amino group, a carboxyl group, a hydrogen, and a side chain group. Amino acids are distinguished
by the properties of their side chains.
Amino acids are the basic structural units of proteins. An amino acid consists of an amine group, carboxyl
group, hydrogen atom, and a side-chain group, all bonded to a central carbon atom. Amino acids are
classified according to the solubility properties and ionizability which they derive from their side-chains.
Single chain proteins generally range from 50 to 1000 amino acids in length. When describing protein
structure, biologists distinguish primary, secondary, tertiary, and quaternary levels of structure. A protein's
primary structure is the actual sequence of amino acids. The secondary structure refers to local bends, kinks
and spirals along the chain. Tertiary structure refers to the shape of the entire polypeptide chain, and
quaternary structure is used to describe proteins which consist of more than one polypeptide chain.
The levels of protein structure.
A protein's state of ionization depends on the nature of its amino acids and the chemical environment. In
neutral, aqueous solution (pH = 7), a protein with a preponderance of basic amino acids, lysine, arginine, or
histidine, will have an overall positive charge.
A protein with many basic side chains will have a positive charge a physiological pH.
Conversely, a protein with many acidic amino acids, glutamic acid or aspartic acid, will have an overall
negative charge in neutral solution.
A protein with many acidic side chains will have a negative charge a physiological pH.
Because the state of ionization depends on the pH of the environment, almost all proteins placed in a basic
environment will accrue a negative charge, losing hydrogen ions as a function of acid/base equilibrium. A
protein placed in an acidic environment will tend to become positively charged. Nondenaturing protein
electrophoresis is generally carried out in a weakly basic environment. In this environment, most proteins
will become negatively charged and migrate towards the positive plate. Denaturing protein electrophoresis,
in the presence of sodium dodecyl sulfate (SDS), also causes proteins to obtain a negative charge through
emulsification by negatively charged dodecyl sulfate ions (see Buffer Additives).
Emulsification by sodium dodecyl sulfate gives proteins a net negative charge. Different proteins in the
same SDS solution are imparted with approximately the same charge to mass ratio, an advantage of SDSPAGE Electrophoresis.
The pH where a protein is electrically neutral overall is a function of the type and number of the protein's
ionizable groups. At this pH, called the isoelectric point (pI) of the protein, it will not migrate in an electric
field. Because the distribution of ionizable groups is different among proteins, they differ in their isoelectric
points. This difference is a powerful tool for electrophoretic separation, used in Isoelectric Focusing.
A protein's state of ionization is determined not only by the the nature of its side chains but also by the pH of
the solution environment. In acidic conditions proteins tend to acquire a net positive charge. In basic
conditions, proteins tend to have a net negative charge. Between these extremes, at a precise value of the pH
called the isoelectric point, the value of which is unique for each species of protein, the most
thermodynamically stable form of the protein has equal numbers of positive and negative charges and does
not migrate in an electric field.
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