BAHAN KULIAH BIOKIMIA POWER POINT BAGIAN 1 /BIOCHEMISTRY POWER POINT LECTURES PART 1 | Karya Tulis Ilmiah

Protein Purification

“A basic comprehension of the

methods described here is

necessary for an appreciation of

the

significance

and the

Protein Isolation

•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.

Methods of solubilization animal cells

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.

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/in

2

forced

through a small hole disrupts cells

Centrifugation

Lysate - broken (lysed) cells- can be separated using differential centrifugation

 RPM - “spun down”

separates by density differences or by size (MW) of particles. Cellular fractionation can separate:

•mitochondria •microsomes •ribosomes

Centrifugation: Units





Nf

V

M

dt

r

d

r

v

s



























₂

1

₂

ln

1

Where:

 = 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 Coefficient)

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 time if S is colored “absorbs light” we can use Beer’s Law.

A =



b

c

c - concentration

-millimolar extinction coefficient

A - absorbance b - path length

T - percent transmittance

Activity Measurements

A = - log % T

For the reaction:

NADH



NAD

+

+ H

-enzyme A b so rb an ce

300 nm 350 nm

NADH

NAD+



A

 Max = 340 nm

oxidized NADH millimolar A   

T min

mg _{mg of protein}

}

= Specific activity

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

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 enzyme

If we remove greens & blues the specific activity increases, however, our total activity remains the same.

If

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:

enz

₁

enz

₂

A B C

NADH NAD+

Use of radioactivity

ATP



ADP + Pi

Separate ATP + Pi + ADP on TLC measure radioactivity

Strategy of Purification

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

Characteristic Procedure •Charge 1. Ion exchange

2. Electrophoresis

3. Isoelectric focusing

•Polarity 1. Adsorption chromatography

2. Paper chromatography

3. Reverse phase chromatography

Characteristic Procedure

•Size 1. Dialysis and ultrafiltration 2. Gel electrophoresis

3. Gel filtration

4. Ultracentrifugation

•Specificity 1. Affinity chromatography 2. Immunopurification

•Solubility 1. Salt precipitation

Ionic Strength

C

i

= the molar concentration of the

i

th species

Zi = it’s ionic charge

1M Na

+

Cl

-

Z = 1 Na

+

Z = 1 Cl

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

1M MgCl

₂

Mg

++

= 1M, and Z = 2

while

Cl

-

= 2M, and Z =1

I = (1 x 2

2

)

Mg

+ (2 x 1

2

)

Cl

= 4 + 2 = 3

Salting out

Use

(NH

₄

)

₂

SO

₄

: it is a Very Soluble salt that does not

harm proteins

.

Solubility of



-lactoglobulin as a function of

Chromatography

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.

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.

Ion Exchange

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}

-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

purification.

Once the protein binds, all unbound proteins are

Paper chromatography

Stationary phase vs.. the Mobile phase

Partitioning between the two phases

Partition coefficient

The more H

₂

O 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.

 

 

A

in

mobile

phase

front

solvent

by the

traveled

distance

substance

by the

traveled

distance



f

R

Materials can be visualized by:

•Radioactivity

•_Fluorescence •_{UV absorbency}

•Stained with one of several dyes Ninhydrin

Iodine

Gel Filtration

Size exclusion

A matrix with holes in it.

Vt = Vx + Vo

Vo = void volume = volume outside the “caves or knooks and crannies”

Ve = elution volume Vo = exclusion volume

Common matrix: dextran, agarose, or polyacrylamide

also desalts 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.

Dialysis is a process that separates molecules according to size

through the use of semipermeable membranes containing

Affinity Chromatography

Based on molecular complementary between an enzyme and

substrate.

The substrate (R) is linked to a matrix with a spacer arm

Electrophoresis

The migration of ions in an electric field

F

_ele

= qE where q is the charge

and E is the electric Field strength

Opposing this is F

_friction

=

vf

where

v

=

velocity of migration

f

is the frictional

force.

qE = vf

f

v

q

E



Separates on charge and size

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

Proteins can be visualized by several methods

Stained with a Dye:

Coomassie blue

Fluorescamine stain for

fluorescence

Silver staining very sensitive

proteins can be labeled with

radioactivity

SDS-PAGE

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

.