pH pH is a measure of the concentration of hydrogen ions in solution. To understand this definition, it’s necessary to look at some of the chemical properties of water. Water has a simple atomic structure. There are two single covalent bonds between an oxygen atom and two hydrogen atoms. The resulting molecule is stable. It has no unpaired electrons, so all the orbitals of the outermost energy level are filled and it has no net charge as an ion would.
However, even though the three atoms share electrons in order to fill their energy levels, the oxygen nucleus, with eight positively charged protons has a stronger attraction for electrons than hydrogen with its sole proton.
As a result, the majority of the time the electrons will be closer to the oxygen nucleus than to the hydrogen nucleus. This gives the oxygen a slightly negative charge and the hydrogen atoms a slightly positive overall charge. These partial charges are much less than the unit charges of ions. This is called a polar covalent bond.
Water is one of the most polar molecules known. Polar molecules are sticky. The partial negative charge at one end of a polar molecule is attracted to the partial positive charge of another polar molecule. This weak attraction is called a hydrogen bond. Water forms a lattice of hydrogen bonds. Occasionally, the pull on the electrons by the oxygen becomes so great that the oxygen actually pulls the electron away from the hydrogen entirely. This process is called ionization. For water, ionization looks like this:
H
2
O

H
+
+ OH
-
Because the positively charged hydrogen ion and the negatively charged hydroxyl ion are attracted to one another, they don’t stay ionized for long. Even though in a glass of pure water only one in 550 million water molecules will be ionized at any one time, the ionization is significant because hydrogen and hydroxyl ions participate in many important biochemical reactions. In pure water, the concentration of hydrogen ions equals 10
–7
M. The pH scale measures the concentration of hydrogen ions in solution, and as a convention, scientists just use the exponent to describe the pH. So the pH of pure water is 7. When water ionizes, you get hydrogen and hydroxyl ions. So, the concentration of hydroxyl ions in pure water is equal to the hydrogen ion concentration (10
-7
M). How do you effect a change in the pH? Disturb the
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equilibrium by adding a compound that can dissociate in water to change the concentration of either H
+
or OH
-
ions.
An acid is a compound that can release H
+
ions in solution. Bases are compounds that can accept H + ions. In practical terms, a lower pH means a higher H + concentration, or greater acidity, and a higher pH means a lower H
+
concentration, or greater alkalinity. It is important to note that the pH scale is logarithmic. A solution with a pH of 5 is 10 times more acidic than one with a pH of 6 (it has 10 times the concentration of H
+
). A solution of pH 4 is 100 times more acidic than one with a pH of 6.
There are also compounds called weak acids and bases. These compounds can ionize in water, but they can also reassociate in water. These compounds play a special role in living systems. They act as buffers to minimize changes in pH by donating or accepting H + or OH ions. A given amount of acid or base causes a smaller change in pH in a buffered solution than in an unbuffered one.
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Proteins
Proteins are the most abundant molecules present in all living cells. If you were to dehydrate an organism’s body more than half of the cellular dry weight would be protein. It is estimated that the typical mammalian cell has at least 10,000 different proteins. Proteins are the macromolecules of the cell that “make things happen.” Proteins determine much of what moves in and out of cells, they regulate the expression of genes, and they form the machinery for biological movement, ranging from muscle contraction to the motion of flagella. Proteins constitute the major structural components of skin, ligaments, tendons and thousands of other structures. Another essential function is the catalysis of biochemical reactions by enzymes.
Every protein has a unique structure and three-dimensional shape. Yet proteins are all composed of only 20 different amino acids. This composition gives us the definition of a protein as a polymer made up of amino acid monomers.
Most amino acids consist of an asymmetric carbon, termed the alpha carbon, which is covalently bonded to:
1.
a hydrogen atom
2.
a carboxyl group
3.
an amino group
4.
an R group: variable side chains specific to each amino acid. The physical and chemical properties of the side chain determines the uniqueness of the amino acid.
3

Amino acids are grouped into four families based on the characteristics of the R group they possess.
1.
Nonpolar (hydrophobic)
2.
Polar, uncharged
3.
Negatively charged (acidic)
4.
Positively charged (basic)
Properties of proteins will be directly related to their specific sequence of amino acids, and this will determine the type of chromatographic technique used to either separate or purify a specific protein.
4

5

Amino acids are ionizable in aqueous solutions. Those that have a single carboxyl group and a single amino group exist at physiological pH (pH 7.4) as the fully ionized zwitterion (see glycine in Figure above).
The types of R groups present add additional ionizable groups (or other characteristics) to a protein. Acidic or basic side chains will be ionized as a function of the pH of the system.
Every protein, at a certain pH called the isoelectric pH or isoelectric point (pI), will contain an equal number of positive and negative charges and will be electrically neutral. At this pH, the protein in its least soluble state in an aqueous solution. At a pH higher than its pI a protein will be negatively charged, and at a pH lower than its pI a protein will be positively charged.
Amino acids are linked together by dehydration synthesis to form polypeptide chains.
The type of covalent bond formed between the linked amino acids is the peptide bond.
Peptide bonds absorb UV light strongly in the 200-220 nm range. Aromatic side chains also absorb in the 250-280 nm range. This property is often exploited to detect and quantitate proteins.
A protein’s function depends upon its unique conformation. A protein is said to be in its native conformation when it is under normal biological conditions. Conformation is a result, not only of external conditions, but also of levels of protein structure that start with the linear sequence of its amino acids (primary structure). The next level of conformation (secondary structure) depends on the bulkiness of the R groups, and leads to formation of

-helices and

pleated sheets which involve hydrogen bonds. The R groups can also interact with each other based on their chemical characteristics. Hydrophobic side chains will interact, and charged groups will interact. If two cysteine residues are in close configuration a disulfide bond can be formed (tertiary structure). Finally, a native protein may consist of more than one polypeptide chain, and this is referred to as quaternary structure. The interaction of the polypeptide chains involves hydrogen bonds and hydrophobic interactions.
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Separation of Proteins
Chromatography is one of the most widely used separation techniques. It is popular for several reasons: 1) separation can be carried out inexpensively, 2) the technique can be mastered in a short amount of time, 3) the resolving power is high for mixtures containing a large population of similar compounds, and 4) methods exist for separating proteins which take advantage of protein size, shape, charge, hydrophobicity, and function.
There are two common characteristics of all chromatographic techniques. First, they are dynamic and cascade processes involving the continual movement of one phase (moving phase) which originally contained the mixture of proteins you wish to separate, relative to an immobile phase (stationary phase). Second, separation is accomplished by the differential movement of the respective proteins in the mixture due to their selective interaction with the stationary phase. The feature that distinguishes one chromatographic procedure form another is the basis for the interaction of proteins with the stationary phase.
 Size (Size Exclusion, Gel Permeation, Gel Filtration Chromatography)
The size of a protein is measured as molecular weight (MW) in daltons. (A dalton is the approximate mass of a hydrogen atom). Size of proteins varies from a few thousand to millions of daltons MW.
The shape or tertiary structure of a protein is important in the separation of mixtures of protein molecules. A compact globular protein will look smaller than an identically sized (MW) protein which is long and stringy.
The stationary phase for this type of chromatography consists of small spherical gel particles which can be regarded as molecular sieves. The gel is obtained by crosslinking chains of carbohydrate, and the size of the pores of the gel is determined by the amount of cross-linking. A large family of gel particles are available which differ in the amount of cross-linking, and, thus, in the size of proteins which they can fractionate.
When a mixture of compounds is applied to the column, molecules will penetrate gel particles if the molecules are smaller than the pore size of the gel. These molecules will progress more slowly through the column due to entrance into and through the gel particles. Molecules which are larger than the pore size of the gel particles can’t penetrate the gel particles and will pass directly through the column.
 Charge (Ion Exchange Chromatography)
Differences in the acidic and basic properties of individual proteins can be exploited to separate mixtures. The overall charge on a protein can be influenced by altering the pH of the solution. If the pH of the solution is raised above the pI of the protein it will be negatively charged overall. If the pH of the solution is lowered below the pI of the protein, the protein will be positively charged overall. In ion-exchange chromatography, the stationary phase consists of small, semi-solid spheres (resins) which contain ionizable groups that can exchange reversibly with other ions in a
7

surrounding fluid. A resin is a cation exchanger when the ionizable group is acidic in nature (interacts with positively charged proteins). A resin is an anion exchanger when the ionizable group is basic in nature (interacts with negatively charged proteins).
Because resin particles are stationary, the effect of the exchange is to retard the movement of ionic solutes through the column. The extent of binding of a given protein will be controlled by the magnitude of the electrostatic force or attraction between the charged protein and the ionizable group on the resin (pH is important!) If a mixture contains a number of proteins each with a different net charge, they will interact to different degrees with the ion exchange resin. Proteins with greater net charge will interact with resin particles to a greater degree and, therefore, will be retarded on the column for a longer time. The resolving power of this technique is very high. The figure below illustrates cation-exchange chromatography.
 Hydrophobicity (Hydrophobic Interaction Chromatography, Reverse Phase Chromatography)
These methods rely on the interaction of hydrophobic protein moieties with the stationary phase resin which contains covalently bonded hydrophobic groups.
Retention time on the column will depend on the number and accessibility of hydrophobic groups for a given protein.
 Function (Affinity Chromatography)
This method involves a binding interaction between an active protein (enzyme, antibody) and the specific substrate or target molecule. The protein against which an antibody was generated (antigen) is bound to resin, and serum from the immunized animal is passed over the column. Antibody molecules recognizing the antigen will be retained on the column, and all other molecules will flow through.
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Protein Electrophoresis (SDS-PAGE)
Electrophoresis is the movement of charged molecules in an electric field. How fast a molecule moves is dependent upon the strength of the electric field, the net charge on the protein, and the medium through which it travels.
Electrophoretic separation of biological macromolecules are almost always performed in gels made of a porous insoluble material such as agarose or acrylamide. For separations involving proteins, acrylamide is the medium of choice because it is inert and, therefore, does not interact appreciably with the samples. Polyacrylamide gels are formed by reacting acrylamide and a cross-linking agent, methylenebisacrylamide (bis), in the presence of an initiator such as
N,N,N’,N’-tetramethyl-ethylenediamine (TEMED) and a catalyst for polymerization (ammonium persulfate). The acrylamide monomers are cross-linked to form a polyacrylamide matrix.
The average distance between the rod-like polymers is referred to as the pore size of the gel. Pore size can be varied by changing the concentrations of acrylamide and the cross-linking agen in the monomer solution (higher acrylamide concentrations result in the formation of smaller pores).
The polyacrylamide gel serves as a molecular sieve that separates proteins on the basis of their size, with smaller proteins migrating more quickly and, thus, farther through the gel. Larger molecules are impeded by the pores and migrate only a short distance.
9

In order to perform separations based solely on size, all of the molecules must have the same charge-to-mass ratio. This can be accomplished by electrophoresing the samples under denaturing conditions, using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
SDS-PAGE is a simple and sensitive method used to fractionate proteins and provide an estimate of their denatured molecular weights. Protein samples are first boiled in a buffer containing SDS, an anionic detergent, and a reducing agent such as

-mercaptoethanol or dithiothreitol, which breaks disulfide bonds and disrupts the tertiary structure of the protein. The negatively charged SDS molecules bind to the hydrophobic amino acid residues disrupting the noncovalent interactions. Proteins bind about 1.4 g of SDS per gram of protein. This translates to about one molecule of SDS per two amino acid residues. This quantity of highly charged detergent molecules is sufficient to overwhelm the intrinsic charge on the protein. Because of the mutual repulsion of the negatively charged sulfate groups on the SDS anions, the protein takes on a linear conformation.
The denatured proteins are then loaded onto a polyacrylamide gel and electrophoresed.
The negatively charged molecules migrate through the matrix toward the anode at a velocity that is roughly proportional to the logarithm of their molecular weight. How fast they travel is dependent on the pore size of the matrix and on the molecular weight of the protein. As the separation is based on differential migration rate, a tracking dye which migrates faster than all the
10

protein components is added to the samples. When this dye, usually Bromphenol blue, reaches the bottom of the gel, the current is turned off.
Visualization of the proteins after electrophoresis is accomplished by first staining with
Coomassie blue, and then destaining with methanol and acetic acid. The protein bands are specifically stained, while the rest of the gel is destained, forming blue protein bands against a clear background.
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