Bio 98 - Lecture 4
Amino acids,
proteins & purification
Tryptophan and Tyrosine absorb UV light at
longer wavelengths than other amino acids
100%
97.2%
Lambert-Beer Law: OD or Abs = log10 Io/I = -log10 I/Io = e c l
e, the molar extinction coefficient, is the optical density (OD) of a material
at a given concentration, c, and l is the pathlength of the cuvette.
The molar extinction coefficient for Trp is 5,500 M-1 cm-1 at 280 nm,
which means a 1 M solution of Trp has an optical density of 5,500 at 280
nm (OD280) when using a pathlength of 1 cm.
Suppose we know that a protein contains 3 Trp and 4 Tyr residues.
What is the extinction coefficient at 280 nm of the protein?
Extinction coeff. (1 M of substance) per Trp is 5,500 M-1cm-1 while
that per Tyr is 1,400 M-1cm-1.
Total extinction coefficient of protein is then
nTrp x eTrp + nTyr x eTyr = eTotal
3 x 5,500 M-1cm-1 + 4 x 1,400 M-1cm-1 = 22,100 M-1cm-1 = e
So a 1 M solution of this protein has an absorbance (Abs or OD) of
22,100 at 280 nm (OD280).

What is the OD280 of a 10 mM solution of this protein if we are
using a cuvette with a pathlength of 1 cm?
e
x
c
x
l
= Abs280
22,100 M-1 cm-1 x 10-5 M x 1 cm = 0.221.
What fraction of the 280 nm photons have not been absorbed after
passing through 1 cm of this sample?
Abs = log10 Io/I = -log10 I/Io or
I/Io = 10-0.221 = 0.60
Isoelectric point (pI)
The isoelectric point (pI) is the pH at which a particular molecule
or surface carries no net electrical charge.
At a pH above their pI, proteins or amino acids carry a net negative
______
charge.
At a pH below their pI, they carry a net positive charge.
AA with non-ionizable side chain (Gly) AA with ionizable side chain (Glu)
pI = 1/2 (pK1 + pK2)
= 1/2 (2.34 + 9.60)
= 5.97
pI = 1/2 (pK1 + pKR)
= 1/2 (2.19 + 4.25)
= 3.22
Peptide: A-N-G-E-L-I-A
Ionizable
group
pK
Charge of group
protonated / deprotonated
Charge at
pH 9.69
Charge at
pH 0
Ala (A)
C-term
2.34
0 / -1
-1
0
Glu (E)
side chain
4.25
0 / -1
-1
0
Ala (A)
N-term
9.69
+1 / 0
+0.5
1
-1.5
1
Net charge
pI = (2.34+4.25) / 2
= 3.30
I. WHY PURIFY PROTEINS?
A. Research - academic
• to study function & regulation
• to determine sequence & 3D structure
• to clone and study the gene
B. Commercial ($$$) – practical
• research tools (restriction enzymes)
• diagnostic reagents (prostate-specific antigen)
• therapeutics (insulin, growth hormones, vaccines,
antibodies [biologics])
II. SOLUBLE vs MEMBRANE PROTEINS
• Proteins must be free in solution for purification.
Not an issue for “soluble proteins.”
• Integral membrane proteins (those imbedded in
the lipid bilayer) must be solubilized with a
detergent first.
Membrane Protein Extraction
Using Detergents
III. STEPS IN THE PURIFICATION OF A TYPICAL
SOLUBLE PROTEIN
1. Homogenization: Prepare cell-free extract (rupture cell
walls/membranes).
2. Centrifugation: Remove membranes, nuclei, large
organelles (mitochondria) etc., keep supernatant.
3. Ammonium sulfate precipitation:
(1) Proteins often become less soluble when the ionic
strength is increased to very high levels. Precipitation
points vary by protein, so purification can be achieved.
(2) Precipitates can be redissolved in a small volume, so
concentration can be achieved.
(3) Dialyze the redissolved proteins against a low salt
buffer.
4. Final purification with one or more rounds of Column
Chromatography
Salt fractionation
Add salt
to 20% saturation
Centrifuge &
remove
supernatant
Centrifuge & remove
supernatant
Add salt to
40% saturation
Cell free extract
Ammonium sulfate (NH4)2SO4
2NH4+
SO4-2
Dialysis lowers salt concentration in a protein solution
before
after
General Chromatography Protocol
OD280
Three Main Types of Chromatography
Ion (anion) exchange
Size exclusion
Know Thy pI
aka gel filtration
Specific affinity
After [NaCl] increase, protein B
will come off the bead before
protein C…
Affinity purification of a genetically engineered (recombinant)
protein containing an engineered purification tag
H
Ni
+5
H
H
H
His-tagged protein:
Engineered, usually 6-His (HHHHHH) at N- or C-terminus
Affinity column or beads: immobilized Ni2+ or Co2+
How does one monitor the purification of a protein?
SDS-PAGE (sodium dodecylsulfate - polyacrylamide gel
electrophoresis)
About 1 SDS molecule binds per 2 amino acids
Activity vs. Specific Activity
A
B
Both beakers have
the same activity
(units) of ‘red’,
though B has a
higher specific
activity as the ratio
of red to other has
increased.
Specific activity =
units/mg
2D Gel (MW vs. isoelectric point)
Ribosome with and without L27 protein