MSc Medical Biochemistry, Ph.D,.
•Must have sensitive method for detection.
•Select a good source for the protein.
a. Rich source of material.
i.e. Heart mitochondria for cytochrome C
b. baker’s yeast (Saccharomyces cerevisiae)
c. Escherichia coli (recombinant expression)
•Tissue specificity: Brain vs. kidney vs. eye.
Chickens, cows, pigs or rats are often used.
Molecular cloning techniques have allowed
biochemists to over-express desired proteins in bacteria or
C.H.O. (Chinese Hamster Ovary) cells by isolating the
gene and placing it into a host system.
METHODS OF SOLUBILIZATION
Cells can be lysed by hypotonic shock.
Cells with high salt inside and no salt outside will
swell and rupture
Bacteria outer membranes must be digested.
•Hen egg white lysozyme digests b (1-4) linkages in
the (glycosidic bonds) of polysaccharides.
Mechanical breakage blenders homogenizers
•French press - high pressure 20,000 lbs/in2 forced
through a small hole disrupts cells
•ultrasound or sonication disrupts cells.
Lysate - broken (lysed) cells- can be separated using
RPM - “spun down”
separates by density differences or by size (MW) of particles.
Cellular fractionation can separate:
= angular velocity
v = velocity of particle
R = distance from center of rotation
M = molecular weight
V = partial specific volume of particle
= density of solvent
Sedimentation velocity (Svedberg
S = s x 10-13
H-bonds, ionic bonds, Van der Waals interactions, and
Hydrophobic interactions can be disrupted.
Denaturation is the process by which a protein loses its
“native” or active shape or conformation.
Temperature can play a role
Protect against-Proteases, Inhibitors, Changes in pH,
Protein can be air-denatured -egg white meringue -
absorption to surfaces
Damaged by oxidation 02
Heavy and transition metals damage proteins -they bind to
protein- Cu+ Hg+
Bacterial contamination can destroy the protein
STABILITY: PROTEINS CAN
In order to follow the purity of an enzyme, you
need a method to measure its activity.
Spectraphotometric analysis- is one common method to
Substrate [S] Product [P] a change of [S] with time
if S is colored “absorbs light” we can use Beer’s Law.
A = eb c
c - concentration
e - millimolar extinction coefficient
A - absorbance
b - path length
T - percent transmittance
A = - log % T
if A then c at max
For the reaction: NADH NAD+ + H-
300 nm 350 nm
Max = 340 nm
mg of protein
} = Specific activity
Volume is 1 ml so micromoles
Start with one liter of lysed cells.
We measure the rate of .01 ml of cells at at concentration of 20
mg/ml. i.e. the amount of enzyme we will assay is 0.01 ml
We get a rate of A = 0.5 A/min
1 millimolar = 6.22 A = e mM
0.5/6.22 = .008 millmolar/min and our assay volume = 1 ml
1 millimolar in a volume of one ml = 1 micromole/ml = mole
C=.008 moles in 1 ml/min = .04 moles
0.2 mg min/mg
Total activity: .04 moles x 20 mg/ml = 0.8 moles / ml
0.8 moles x 1000 ml = 800 moles in 1 liter of cells
Red = is our enzyme
If we remove greens & blues the specific activity increases,
however, our total activity remains the same.
We lose red the total activity decreases.
We usually monitor both the total activity and specific activity
for each purification step.
Until the Specific Activity reaches a maximal value.
How do we know if it is pure? Usually SDS - Page
See Table 5-4 in Voet and Voet
Some enzymes have no easy assay but the product of the reaction can be
used in another reaction:
A B C
Coupled Reactions: We couple enz2 to enz1 and measure NADH to get
USE OF RADIOACTIVITY
ATP ADP + Pi
Separate ATP + Pi + ADP on TLC measure radioactivity
Phosphoimager makes this easy else cut spots and count in
STRATEGY OF PURIFICATION
Fractionation procedures or steps to isolate protein based on
•Charge 1. Ion exchange
3. Isoelectric focusing
•Polarity 1. Adsorption chromatography
2. Paper chromatography
3. Reverse phase chromatography
4. Hydrophobic interaction
•Size 1. Dialysis and ultrafiltration
2. Gel electrophoresis
3. Gel filtration
•Specificity 1. Affinity chromatography
•Solubility 1. Salt precipitation
2. Detergent solubilization
Ci = the molar concentration of the ith species
Zi = it’s ionic charge
1M Na+ Cl- Z = 1 Na+
Z = 1 Cl-
1 = (1M x 1)Na + (1M x 1)Cl
For di- or tri-valent ions, where I is different than M
Mg++ = 1M, and Z = 2
while Cl- = 2M, and Z =1
I = (1 x 22)Mg + (2 x 12)Cl = 4 + 2 = 3
Use (NH4)2 SO4 : it is a Very Soluble salt that does not
Refer to the Hofmiester Series
SOLUBILITY OF CARBOXY-HEMOGLOBIN AT
ITS ISOELECTRIC POINT
SOLUBILITY OF B-LACTOGLOBULIN AS A
FUNCTION OF PH
Analytical methods used to separate molecules.
Involves a mobile and a stationary phase.
•Mobile phase is what the material to be separated is
•Stationary phase is a porous solid matrix which the
mobile phase surrounds.
•Separation occurs because of the differing
chemistries each molecule has with both the mobile
and stationary phase.
•Chemistries are different depending on the specific
TYPES OF CHROMATOGRAPHY
•Gas - Solid: Mobile phase is gaseous, stationary phase is
a solid matrix.
•Liquid - Solid: Mobile phase is liquid, stationary phase is
a solid matrix.
• If separation is based on ionic interaction the method is called
Ion Exchange chromatography.
•If separation is based on solubility differences between the
phases the method is called adsorption chromatography.
•If the separation is base on size of molecule the method is
called gel filtration or size exclusion.
•If the separation is base on ligand affinity the method is called
A solid matrix with a positive charge i.e. R+ can bind
different anions with different affinities.
•We can swap one counter ion for another
(R+A-) + B- (R+B-) + A-
R = Resin and exchanges Anions (-)
•This is an anion exchange resin.
•There are also cation exchange resins. The type of an R group
can determine the strength of interaction between the matrix, R
and the counter ion.
• If R is R-
(R-A+) + B+ (R-B+) + A-
PROTEINS HAVE A NET CHARGE.
The charge is positive below pI,
while the charge is negative above pI
The choice of exchange resin depends on the charge of
the protein and the pH at which you want to do the
Once the protein binds, all unbound proteins are
washed off the column. Bound proteins are eluted by
increasing the ionic strength, changing the counter ion
or changing the pH altering the charge on the protein
or the column.
Stationary phase vs.. the Mobile phase
Partitioning between the two phases
The more H2O soluble the slower it migrates.
The more organic soluble the more it migrates.
The aqueous component of the solvent combines with
the cellulose of the paper and becomes the stationary
Materials can be visualized by:
•Stained with one of several dyes
A matrix with holes in it.
Vt = Vx + Vo
Vo = void volume = volume outside the “caves or knooks
Vx occupied by gel beads
Vo 35% of Vt
Ve = elution volume Vo = exclusion volume
Common matrix: dextran, agarose, or polyacrylamide
also desalts proteins
Gel filtration can be used to determine
the molecular mass of proteins
Before swelling the dry bead size 5% of Vt
60% are “holes”
Hole sizes can be made different
Small molecules see a larger column volume
than big molecules and they get hung up in the
Large proteins are excluded, while small protein
Separation on size and shape.
Dialysis is a process that separates molecules according to size
through the use of semipermeable membranes containing
pores of less than macromolecular dimensions
Based on molecular complementary between an enzyme and
The substrate (R) is linked to a matrix with a spacer arm
Only protein that binds R will stick to column. put citrate on column
citrate dehydrogenase will specifically bind. Add excess citrate and
the enzyme will be released.
The purification of Staphylococcal nuclease using
the ligand, diphosphothymadine
The migration of ions in an electric field
Fele = qE where q is the charge
and E is the electric Field strength
Opposing this is Ffriction = vf where v =
velocity of migration f is the frictional
qE = vf f
SEPARATES ON CHARGE AND SIZE
pH matters as well as the pI of the protein.
Can be run at several pH values depending
DNA can also be separated on agarose
gels. Genomic sized DNA can also be
separated but requires more sophisticated
PROTEINS CAN BE VISUALIZED BY SEVERAL
Stained with a Dye: Coomassie blue
Fluorescamine stain for
Silver staining very sensitive
proteins can be labeled with
and visualized by exposure to X-
Add sodium dodecyl sulfate, a 12 carbon detergent to give
a negative charge to the protein.
SDS also denatures the protein and collapses into a
The proteins are separated by molecular mass