PROTEIN PURIFICATION AND ANALYSIS
Basic/initial ste ps
1. Acquire/prepare tissue (1 g = 106 plant cells, 10 9
animal cells, 10 12 bacterial cells)
2. Suspend in buffer (pH stabilizers: phosphate,
Tris/TRIZMA, ÒGoodÓ buffe
rs, e.g. HEPES), with
solubilizers/detergents (e.g. digitonin, Triton X-100),
protease inhibitors: cold, PMSF, leupeptin,
chymostatin)
3. Lyse cells (Waring blendor, sonicator, French
press, mortar and p estle)
4. Centrifuge (e.g. 12 kg x 30 min)
lipid
supernatant =
Òcrude extractÓ
pellet
5. (NH 4)2SO4 or pH precipitations: (Òsalting outÓ):
Salt competes for water of solvation, precipitating
proteins: different proteins precipitate at different
concentrations (e.g., 20%, 50%, 80% of saturation);
2-3-fold purification
Assays
Need measures for the object (enzyme activity, chromophore, etc.)
and for total protein concentration:
Enzyme: A + B --> C + D: detect C (or A) as
func tion of time
(ideally, linear with time and amount of enzyme)
C or D
amount
of substrate
or product
A or B
Time
Spectrophotometry (for activity, protein concentration,
etc.)
A = - log10 (I/I o)
A = a*c*l
a = absorbance (specific for compound);
c = concentration;
l is cuvette thickness (usually 1 cm)
dI/I = -ec dl
ºdI/I = -ecl
lne(I/Io) = -ecl
a = e/2.3
log10(I/Io) = -acl
A = -acl = -log10(I/Io)
Use spectrophotometry for:
1.
2.
3.
4.
Measuring substrate or product concentration if one is
“colored” (absorbs UV or visible light).
Measuring substrate or product concentration if one reacts
with something to form a colored compound.
Measuring the concentration of total protein (by UV
absorbance: A280 ~ 1 @ 1 mg/ml) or of a specific protein (like
hemoglobin) that contains a colored “chromophore.”
Measuring the concentration of protein by reacting it with
a reagent to form a colored compound. (E.g., cys, tyr, trp
reduce Cu2+ to Cu+, which forms an intense blue with BCA;
Coomassie reagent turns blue in hydrophobic environment.)
Measuring purification
Specific activity (SA) = total enzyme activity/total protein or
activity concentration/protein concentration
1 IU (international unit) = 1 µmol substrate used (product formed)/min-mg protein
“Purification” at step x = SA of step x / SA of crude extract
One measure of “purity” is constant SA.
Note: if the protein of interest is 0.2% of the total, then you require 500-fold
purification for purity; if each step gives 3-fold purification, then you need 5-6 steps;
if each step causes some loss of the protein of interest (e.g. 50%), you need to start
with 30-60-times more enzyme than you will recover.
Purification techniques: size, charge, specific binding
Size: exclusion
chromatography
Size
SDS - PAGE
(sodium dodecylsulfate - polyacrylamide gel
eletrophoresis)
SDS: charged detergent that denatures protein and
gives uniform surface charge density
acryla mide gel: cross-links produce resistance to
protein movement
negative electrode
larger protein
smaller pro tein
positive electrode
Isoelectric electric focusing (IEF): separate by pH at
which proteins have zero charge
Use ÒampholytesÓ to stabilize pH gradient in an
electrophoresis gel.
Electrophorese: protein moves to pI (isoelectric point,
zero charge)
negative electrode
high pI (e.g. 10)
low pI (e.g. 4)
positive electrode
2-D gels combine isoelectric focusing with SDSelectrophoresis
Two-dimensional gel electrophoresis: IEF followed by SDS-PAGE
3
Isoelectric point
5
7
9
MW (kD)
150
100
50
25
10
Affinity chromatography: separate by biological specificity
1.
2.
3.
4.
Attach “ligand” to insoluble column material
Combine protein mixture with column: enzyme binds to ligand
Wash column to remove unwanted material
Elute enzyme with substrate or change in pH (to reduce binding)
What do you use for ligand?: su bstrate analog; dyes
simulate enzyme cofactors (often planar, aromatic)
Summary
Enzyme purification involves: breaking cells and stabilizing pH, proteolysis,
measuring enzyme activity and protein concentration,
a series of separation techniques
Spectrophotometry is useful for measuring protein concentration and,
often, enzyme activity
Exclusion chromatography, electrophoresis, isoelectric focusing, and
affinity chromatograph are useful methods for purifying proteins