Bio 98 - Lecture 4
Amino acids, proteins & purification
Tryptophan and Tyrosine absorb UV light atlonger wavelengths than other amino acids
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.
Lambert-Beer Law: OD or Abs = log10 Io/I = -log10 I/Io = c l
97.2%100%
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 Trp + nTyr x Tyr = Total
3 x 5,500 M-1cm-1 + 4 x 1,400 M-1cm-1 = 22,100 M-1cm-1 =
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 M solution of this protein if we are using a cuvette with a pathlength of 1 cm?
xc 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 ______ charge.
At a pH below their pI, they carry a net positive charge.
pI = 1/2 (pK1 + pK2) = 1/2 (2.34 + 9.60) = 5.97
AA with non-ionizable side chain (Gly)
pI = 1/2 (pK1 + pKR) = 1/2 (2.19 + 4.25) = 3.22
AA with ionizable side chain (Glu)
At a pH above their pI, proteins or amino acids carry a net negative charge.
Peptide: A-N-G-E-L-I-A
Ionizable group
pK Charge of group
protonated / de-protonated
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
Net charge -1.5 1
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 ExtractionUsing 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
Cell free extract
Add salt to 20% saturation
Centrifuge & remove
supernatantAdd salt to
40% saturation
Centrifuge & removesupernatant
Salt fractionation
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 Specific affinity
Know Thy pI aka gel filtration
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
HH
H
Ni+5
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
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
A B
2D Gel (MW vs. isoelectric point)
Ribosome with and without L27 protein
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