M.Prasad NaiduMSc 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.

Cells can be lysed by hypotonic shock.

Cells with high salt inside and no salt outside willswell and rupture

Bacteria outer membranes must be digested.Gram-negative bacteria•Hen egg white lysozyme digests (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.

Centrifugation

Lysate - broken (lysed) cells- can be separated usingdifferential centrifugation

RPM - “spun down”separates by density differences or by size (MW) of particles.

Cellular fractionation can separate:

•mitochondria•microsomes•ribosomes•soluble proteins

Centrifugation: Units

Nf

VM

dt

rd

r

vs

1ln122

Where: = angular velocityv = velocity of particleR = distance from center of rotationM = molecular weightV = partial specific volume of particle = density of solventSedimentation velocity (Svedberg Coefficient) 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

“cold labile”“heat labile”

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

In order to follow the purity of an enzyme, you need a method to measure its activity.

Spectraphotometric analysis- is one common method to measure activity.

Substrate [S] Product [P] a change of [S] with timeif S is colored “absorbs light” we can use Beer’s Law.

A = b c c - concentration-millimolar extinction coefficientA - absorbance b - path lengthT - percent transmittance

A = - log % T

if A then c at max

For the reaction: NADH NAD+ + H-

enzyme

Ab

sorb

ance

300 nm 350 nm

NADH

NAD+

A Max = 340 nm

oxidized NADH millimolarA

T minmg mg of protein

} = Specific activity

Volume is 1 ml so micromoles NADH oxidized

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 = 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 ml min

Red = is our enzymeIf we remove greens & blues the specific activity increases,

however, our total activity remains the same.If

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:

enz1 enz2

A B C

NADH NAD+

Coupled Reactions: We couple enz2 to enz1 and measure NADH to get A

ATP ADP + Pi

Separate ATP + Pi + ADP on TLC measure radioactivity

Phosphoimager makes this easy else cut spots and count in scintillation counter.

Pi

ATP

N

NN

N

NH2

O

HOH

HH

HH

OP-O

O-

O

OP-O

O-

O

O-OP-O

O-

O

Fractionation procedures or steps to isolate protein based on physical characteristics.

Characteristic Procedure•Charge 1. Ion exchange

2. Electrophoresis3. Isoelectric focusing

•Polarity 1. Adsorption chromatography2. Paper chromatography3. Reverse phase

chromatography4. Hydrophobic interaction

Characteristic Procedure

•Size 1. Dialysis and ultrafiltration2. Gel electrophoresis3. Gel filtration4. Ultracentrifugation

•Specificity 1. Affinity chromatography2. Immunopurification

•Solubility 1. Salt precipitation2. Detergent solubilization

Ci = the molar concentration of the ith speciesZi = it’s ionic charge

1M Na+ Cl- Z = 1 Na+

Z = 1 Cl-

1 = (1M x 1)Na + (1M x 1)Cl

2

2iZc

2

1 I

i

For di- or tri-valent ions, where I is different than M

1M MgCl2

Mg++ = 1M, and Z = 2

while Cl- = 2M, and Z =1

I = (1 x 22)Mg + (2 x 12)Cl = 4 + 2 = 3 2 2

Use (NH4)2 SO4 : it is a Very Soluble salt that does not harm proteins.

Refer to the Hofmiester Series

Analytical methods used to separate molecules. Involves a mobile and a stationary phase.

•Mobile phase is what the material to be separated is dissolved in.

•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 method.

•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 Affinity chromatography.

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-

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 purification.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 phasePartitioning between the two phases

Partition coefficientThe 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 phase.

phase mobilein A

phase stationaryin ApK

frontsolvent by the traveleddistance

substance by the traveleddistancefR

Materials can be visualized by:

•Radioactivity

•Fluorescence

•UV absorbency

•Stained with one of several dyes

Ninhydrin

Iodine

Sulfuric acid

A matrix with holes in it.

Vt = Vx + VoVo = void volume = volume outside the “caves or knooks and crannies”Vx occupied by gel beadsVo 35% of Vt

Ve = elution volume Vo = exclusion volumeCommon 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 caves.

Large proteins are excluded, while small protein are included.

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 substrate.

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

Electrophoresis

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 force.

qE = vf f

v q

E

pH matters as well as the pI of the protein.

Can be run at several pH values depending on proteins.

DNA can also be separated on agarose gels. Genomic sized DNA can also be separated but requires more sophisticated equipment.

Stained with a Dye: Coomassie blue

Fluorescamine stain for fluorescence

Silver staining very sensitive

proteins can be labeled with radioactivity

and visualized by exposure to X-ray film

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 globular ball.

The proteins are separated by molecular mass