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2. BUFFERS, pH, PROTEIN ANALYSIS & PURIFICATION

Note: this is a more detailed version oI the inIormation given in the syllabus.

It is important Ior cellular Iunction that the pH oI the internal environment be kept constant.
Low blood pH, called acidosis, and high blood pH, called alkalosis, is associated with a number
oI disease states. Compounds that resist changes in pH are called buIIers. The 3 major
physiological buIIers are bicarbonate, phosphate, and proteins. In this lecture, the concept oI
pH is reviewed and the action oI buIIers described. The response oI the amino acids and proteins
to pH is also described in terms oI their identiIication and puriIication.

I. ACID-BASE CHEMISTRY

A. The Henderson-Hasselbalch Equation

An acid is a substance that can donate a proton (H

) in solution. A base accepts protons

and removes them Irom solution. Strong acids, such as HCl, and strong alkalis, such NaOH,
essentially completely ionize in aqueous solution. The dissociation oI HCl in solution can be
represented as HCl H

Cl
-
. The corresponding equilibrium constant, Ka |H

| |Cl
-
|/|HCl|
is extremely large (~10
14
), since there is very little HCl leIt in solution. The H

ion concentration
is taken to be the same as the amount oI acid dissolved in solution.


Dissolving HCl in solution will thereIore acidiIy the solution and lower the pH. The pH is
equal to the negative log (base 10) oI the hydrogen ion or proton concentration ( - log
10
|H

|).
At neutral pH, pH 7, |H

| 10
-7
M. ThereIore, as the H

concentration rises, the pH will drop.

Furthermore, a change oI 1 pH unit corresponds to a 10 Iold change in H

concentration.
Physiological (blood) pH is 7.4 and pH oI other tissues varies (the interior oI the stomach, Ior
example, is about pH 1.5).

The dissociation oI NaOH in solution can be represented as NaOH Na

OH
-
.
Dissolving a strong alkali in water (e.g. NaOH) makes the solution more basic and raises the pH.

Water itselI ionizes into hydrated H

and OH
-
, with the Iollowing relationship:

K
w
|H

| |OH
-
| 1.0 x 10
-14
(at 25 C).

ThereIore, the concentrations oI H

and OH
-
are reciprocal. II the concentration oI one is
known, the other can be calculated. For example, iI |H

| 10
-3
M, then |OH
-
| 10
-11
M.

A strong acid is an acid Ior which the conjugate base holds a proton weakly (gives up the
proton readily). A weak acid (HA) is an acid in which the conjugate base (A) holds a proton
tightly. Here the equilibrium constant is much smaller than that Ior HCl or NaOH.

Ka |H

| |A
-
|/|HA|.

Analogous to writing pH -log|H

|, we can write pKa -log Ka.

The Henderson-Hasselbalch equation is derived Irom rearranging the equilibrium equation.


2
pH pKa log |A
-
|/|HA|.


II. THE ISOELECTRIC POINT

A. Isoelectric Focusing of Amino Acids

Being both an acid and an amine, amino acids have at least two titrating groups. A
monoamino monocarboxylic acid such as glycine, Ior example, has two pKa values denoted
pKa1 and pKa2.

pKa1 pKa2

NH
3
CHCH
3
COOH

NH
3
CHCH
3
COO
-
NH
2
CHCH
3
COO
-

-H

-H

Its titration curve appears in Fig. 3, with the predominant Iorms boxed in:






















At low pH, an amino acid is considered to be Iully protonated (i.e., it carries all the hydrogen
ions possible). As the pH rises, protons
are lost Iirst Irom the carboxylic acid and
then Irom the protonated amine. At
higher pH values molecules become
more negatively charged. Note that
amino acids can exist as a doubly charged
ion with no net charge. This is called a
zwitterion. Most amino acids exist
predominantly as zwitterions in the body.
At pH equal to pKa1, halI an amino acid
is in the

NH
3
CHRCOO
-
Iorm and halI
in the

NH
3
CHRCOOH Iorm. At pH
equal to pKa2, halI is in the zwitterion
Iorm and halI in the NH
2
CHRCOO
-
Iorm.
+
-
pH 1 (Alanine is positively charged)
pH 6 (Alanine is about neutral)
pH 11 (Alanine is negatively charged)
Apply amino acid here
Spot will migrate in
electric field until neutral

Isoelectric focusing.

NH CHRCOOH
NH CHRCOOH 3
+
NH CHRCOO
-
3
+
NH2CHRCOO
-

3
+
NH CHRCOO
-
3
+
pKa1=2.34
NH CHRCOO
-
3
+
NH CHRCOO
-
2
pKa1=9.69
pI=6.02
R = CH3
12
10
8
6
4
2
0
0.5 1.0 1.5 2.0
Equivalents OH
pH
-

The dependence of the charges of an amino acid on pH.

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A molecule with no net charge will not move in an electric field. The pH at which an
amino acid is electrically neutral is called the isoelectric point (pI) and is given by: pI (pKa1
pKa2)/2 Ior an amino acid with only 2 titratable groups. In isoelectric Iocusing, a stable pH
gradient is built up between two electrodes. An amino acid will travel to the point where it is
isoelectric and stop. The isoelectric pH value is a useIul property Ior identiIication oI the amino
acid.

B. Isoelectric Focusing of Proteins

Now consider the acid-base properties oI a peptide or protein (Fig. 5). Note that the
Iormation oI the peptide bond between the amino and a carboxyl group has eliminated their
acid-base properties. The most amino terminal amine and the most carboxyl terminal carboxyl
groups are still present. The only other acid-base properties oI a protein are contained in the R-
groups.

+
H
3
N C CO
2
-
H
R
1
+
H
3
N C CO
2
-
H
R
2
C
H
C
H
C
R
1
N
H
O
CO
2
-
R
2
+
H
3
N + H
2
O +
Peptide bond

Fig. 5 Formation of the peptide bond. The carboxyl group of one amino acid combines with the amino
group of another. Water is eliminated during the reaction.




Consider the peptide in Figure 6,
which, like most proteins, has more than
two titrating groups. The titration curve is
more complex than a single amino acid.
Furthermore, the isoelectric point oI this
peptide (positively charged at pH 7) is
diIIerent Irom that oI a neutral peptide or
amino acid (no ionizable group in the
side-chain).


The ionization oI this peptide has several steps:

CO
2
H
net
charge:
pKa1
+2
pI = (pKa2 + pKa3)/2 = 8.05
+
H
3
N
CO
2
- +
H
3
N
CO
2
- +
H
3
N
CO
2
-
H
2
N
pKa2 pKa3
0 -1
NH
NH
+ NH
N
+1
NH
N
NH
NH
+
His
0
His
0
His
+
His
+
2.6 6.5 9.6
... ... ... ...

+
H
3
N CH
CH
3
C
O
N C
H
2
C
O
N CH C
O
N
CH
2
CH COO
-
H
2
C
CH
H
3
C
H H
H
NH
H
N
+
CH
3

Structure of the tetrapeptide
alanyl-glycyl-histidyl-leucine (at pH 7).

4
+
+
+
+
+
+
+
+
NH ... His ... COO
NH ... His ... COO
NH ... His ... COO
NH ... His ... COO
NH ... His ... COO
2
4
6
0.5 1.0 1.5 2.0
8
10
12
pH
3.0 2.5
+ -
NH ... His ... COO
3
+
NH ... His ... COOH
3
pKa1=2.6
+
NH ... His ... COOH
3
+ -
NH ... His ... COO
3
pKa2=6.5
+ -
3
-
3
o
-
NH ... His ... COO
3
o
-
2
o
-
3
o
pKa3=9.6
-
2
o

Titration of a tetrapeptide, alanyl-glycyl-histidyl-leucine. Only ionizable groups are represented. His

and His
o

represent the protonated and unprotonated Iorms, respectively, oI the histidine side-chain.

Note the isoelectric point is higher than in the amino acid alaninethis peptide migrates to a
diIIerent position (pH 8.05) in isoelectric Iocusing.

Isoelectric Iocusing is a powerIul technique not only Ior puriIying, but also Ior identiIication
oI amino acids, peptides, proteins, and other molecules. It is one oI several methods Ior
detecting and analyzing proteins.

III. PROTEIN IDENTIFICATION

Procedures Ior identiIying proteins are based on their physical and chemical properties
such as solubility, charge, size and shape, and binding speciIicity. The surIace groups on
proteins are largely responsible Ior a protein's solubility, net charge, and speciIicity. The
acid-base behavior oI these surIace groups is particularly important.

Analytic methods usually are more precise Ior the characteristic property oI the protein and
also usually have higher sensitivity. Some oI these methods destroy the protein.

1. Analysis based on charge

Electrophoresis is usually carried out in a solid support gel composed oI agarose or

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polyacrylamide. An electric Iield is set up between two electrodes. Cations (molecules in which
the net charge is positive) move toward the negatively charged cathode, and anions (molecules in
which the net charge is negative) move toward the positively charged anode. The major Iactor is
the net charge. The greater the net charge the Iaster the molecule will migrate (although size and
shape also inIluence the rate oI migration).

The net charge oI a protein can be calculated because pKa values Ior the protein's amino acid
side-chains can be estimated at a given pH. As discussed above, there is a pH value Ior every
protein at which it has no net charge, the isoelectric point (pI), and will thereIore not move in an
electric Iield. In isoelectric Iocusing a protein will migrate to the pH corresponding to its pI.

2. Analysis based on molecular size
a. Electrophoretic analysis under denaturing conditions can be used to determine the purity and
size oI individual protein. Proteins are denatured by heating in the presence oI -
mercaptoethanol and the negatively charged ionic detergent, sodium dodecyl sulIate (SDS). One
SDS molecule binds Ior about every two amino acids oI the protein. The denatured proteins,
being coated with SDS and hence negatively charged, are then separated on the basis oI their
size (not their charge) by SDS-PAGE (polyacrylamide gel electrophoresis) on gels that contain
SDS. Smaller proteins migrate through the pores oI the gel faster. The proteins are stained Ior
visualization.


b. Sedimentation. Under high centriIugal Iorces, a molecule sediments at a rate characterized
by the sedimentation coefficient, S. The sedimentation rate depends on the molecule's size,
shape and density, and on the density and viscosity oI the solvent.

c. Mass Spectrometry. The mass oI a protein (as well as smaller molecules) may be accurately
determined on a mass spectrometer.


3. Amino acid composition and sequencing of proteins

Sometimes one wishes to determine the composition oI a protein to assess the nutritive value
as some proteins are deIicient in particular amino acid residues. The protein is cleaved into its
constituent amino acids typically by heating the protein in 6M hydrochloric acid Ior 24 hours.
Amino acid analyzers are devices Ior determining the amino acid composition, use column
chromatography Ior separating the amino acids. The amino acids are quantiIied by reaction with
a chemical (ninhydrin), which produces a highly intense blue color (except Ior proline).

The amino acid sequence is sometimes used to conIirm protein identity. Most commonly,
this is done by deduction oI the amino acid using the known DNA sequence. Protein sequences
are determined directly by mass spectroscopy or by automated protein sequencers using the
Edman Degradation reaction. Each method has limitations. In particular, the Edman
Degradation method works only on relatively short peptides oI less than 50 residues. The
strategy Ior peptide chains oI more than 50 residues is usually to break the chain into a small
number oI Iragments by internal cleavage at speciIic amino acid residues, separate the Iragments
and determine the sequence oI each peptide. Using proteolytic agents oI diIIering speciIicity,
small peptides with overlapping sequences may be obtained and the sequence oI the entire
protein deduced. The site oI cleavage oI a polypeptide may be on either side oI an amino acid:

6
R
N-side HN-CH-CO C-side

Two proteolytic enzymes, trypsin and chymotrypsin, and one reagent, cyanogen bromide, are
commonly used to cleave large polypeptides.



Enzyme or reagent PreIerred site oI cleavage

Trypsin C-side oI Lys and Arg (basic amino acids, except his)
Chymotrypsin C-side oI Phe, Trp, Tyr (aromatic amino acids)
Cyanogen bromide (CNBr) C-side oI Met

The Edman degradation reaction reaction modiIies only the N-terminal amino acid oI a
chain. The modiIied amino acid (the phenylthiohydantoin) can be selectively cleaved Irom the
chain using hydroIluorous acid (HF) and identiIied. Its cleavage exposes a new N-terminal
amino acid residue, which is then identiIied in subsequent cycles oI modiIication and cleavage.
The process requires only picomole quantities oI protein.


N
C
S
Phenylisothiocyanate
+
OC
CH R
NH
2
NH
C S
rest of protein
CH R
HN
N
NH
S
O
R
Phenylthiohydantoin
rest of protein
addition release

rest oI protein
Synthesis scheme Ior Iorming a derivative oI the N-terminal amino acid Ior identiIication. Each
cycle shortens the peptide by one amino acid residue.


Summary of Amino Acid Properties

Name Side-chain Properties
Alanine hydrophobic
Arginine positively charged at pH 7
Asparagine side-chain resembles a peptide bond (polar)
Aspartate negatively charged at pH 7; pKa ~4
Cysteine capable oI Iorming disulIide bonds; pKa ~8
Glutamate negatively charged at pH 7; pKa ~4
Glutamine side-chain resembles a peptide bond (polar)
Glycine small side-chain allows close approaches and sharp bends
Histidine (Partial) positive charge at pH 7; pKa ~6
Isoleucine hydrophobic
Methionine hydrophobic
Lysine positively charged at pH 7

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Leucine hydrophobic
Phenylalanine hydrophobic; larger than alanine; UV absorbance
Proline cyclized, hydrophobic, helix breaker
Serine polar
Tyrosine (mostly) hydrophobic; UV absorbance
Tryptophan (mostly) hydrophobic; UV absorbance
Valine hydrophobic
Threonine (mostly) polar


IV. PROTEIN PURIFICATION

Most oI the techniques used to identiIy proteins can be optimized to produce large quantities
oI Iunctional proteins. The key to understanding the mutated Iorms oI p53 described in the
previous lecture, involved puriIication oI each protein mutant and studying its Iunction. The Iirst
step oI protein puriIication is usually homogenization (breaking up) oI the tissue or source that
contains the protein of interest. To obtain the protein myoglobin the appropriate tissue would
be muscle. The domains oI the p53 protein, Ior example, can be expressed in a recombinant
system, using techniques similar to those described in Lecture 7.

1. Separation based on solubility

The homogenate is centriIuged to remove large particles, such as broken cell walls and large
membrane Iragments. The crude protein mixture is now ready Ior puriIication.

A protein has limited solubility in a given salt concentration, solvent, pH and temperature.
Varying these Iactors can alter the solubility and can thus be used Ior protein separation.
a. Most proteins are least soluble at their isoelectric point. The technique is called
isoelectric precipitation.

b. Temperature: Increasing temperature Iirst increases solubility and then causes unfolding,
oIten with decreased solubility. Proteins have diIIerent tendencies to denature, i.e., they
denature at diIIerent temperatures and may be separated by heating to a particular temperature.

c. Salt concentration: The solubility oI globular proteins is usually increased by the presence
oI salts, with maximal solubility near physiological ionic strength (about 0.2 M). The procedure
is called salting-in. They may be precipitated by removal oI the salts by dialysis through use oI a
semi-permeable membrane that allows the salts, but not the large proteins, to escape. On the
other hand, some proteins are precipitated out oI solution by high concentrations oI neutral salts
(usually ~ 1 M). The mechanism is unclear. The most commonly used salt is ammonium sulIate.
Salting-out is most eIIective near the isoelectric point oI the protein.

d. Solvent: Water-soluble proteins may be precipitated Irom aqueous solution by adding
miscible organic solvents such as ethanol. Membrane proteins usually require a detergent (soap)
Ior solubilization.



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2. Separation based on protein charge

Ion-exchange chromatography employs synthetic resins consisting oI a neutral insoluble
support to which ionizable groups (either negatively or positively charged) have been attached.
pH changes the charges on the protein (and sometimes the column) thus changing the aIIinity. In
addition, high salt concentrations decrease the interaction between protein and column material.

3. Separation based on size

a. In gel filtration chromatography, a solution oI protein is placed on a column packed with
tiny beads oI a highly hydrated, cross-linked polymeric material (a gel). Proteins oI diIIering size
vary in their ability to penetrate the pores oI the beads. The smaller the protein, the higher the
probability that it will enter the beads and thus, its passage down the column is retarded. Large
proteins that cannot enter the gel beads pass through in a smaller volume. The order oI
emergence Irom the column is opposite to that oI the SDS-PAGE gel. Gel Iiltration is also used
to estimate the molecular weights oI puriIied proteins and thereIore is also an analytical method.

b. Dialysis. Semi-permeable membranes exist that permit small molecules to pass through them,
but that are impermeable to larger molecules. These semi-permeable membranes can be used to
separate small molecules (such as salts and metabolites) Irom larger proteins.

4. Separation based on selective interactions with other molecules

Some proteins such as enzymes, immunoglobulins, and receptor proteins have binding sites
designed to recognize another substance and bind very speciIically to this substance (or other
molecules that structurally resemble it). Advantage can be taken oI this speciIic binding aIIinity
in puriIying these proteins. In general, the procedure is to attach the recognized substance
covalently to an insoluble resin. From a mixture oI proteins eluted through the resin, the protein
oI interest will be selectively retarded. Affinity chromatography is a very eIIective technique
and generally yields more highly puriIied proteins than most other techniques. For example, the
DNA-binding protein, p53, may be puriIied by application to a column that has covalently
attached DNA. p53 is eluted by disrupting the protein-DNA interaction with high salt, changing
the pH, or adding excess DNA molecules.

Antibodies are sometimes used to puriIy proteins. An antibody is a protein produced by an
animal in response to the presence oI an antigen-generating substance called an antigen.
Antibodies can be raised against a small amount oI a puriIied protein. The antibody can itselI be
puriIied and covalently attached to a column resin while leaving the antigen-binding sites open.
II more protein is to be puriIied, the antibody column can be used to separate the protein Irom a
complex mixture. The protein-antibody interaction is the basis oI the Western Blot which is
mostly used as an analytical method to determine the identity and sometimes the quantity oI a
protein.


Internet Links:
BuIIers and pH: www.wiley.com/college/pratt/0471393878/student/review/acidbase/index.html
Protein puriIication: http://www-users.med.cornell.edu/~jawagne/proteins&puriIication.html
Structure-based drug design: pubs.acs.org/cen/coverstory/7923/7923drugdesign.html