BS(integrated) BIOCHEMISTRYEmail ID:khair_awkum@yahoo.com

BiochemistrySixth Edition

By Khair Ullah

Chapter 3:Exploring Proteins and Proteomes

Methods in Protein Chemistry

These are methods used in isolation, purification, detection, degradation, analysis and synthesis of proteins.

As one would expect, most of these involve aqueous media and require a knowledge of pH, pKas, and charge on a peptide at various pH values.

Proteome: defines the compete functional information about a group of proteins thatwork together as a functional unit.

Assay of an Enzyme

In this reaction, the increase in absorbance of NADH at 340 nm is used to follow the formation of pyruvate

This is an oxidation-reduction reaction

Enzyme Activity

Total activity in soution use Unit of enzyme activity: µmol substrate/min or mol S/sec = katal

To follow during purification use Specific activity: µmol substrate/min-mg E

or mol S/sec-kg E = katal/kg E

To compare different enzymes use Molecular activity also called turn-over number (TON): µmol substrate/min-µmol E

or mol S/sec-mol E = katal/mol E

Centrifugation

Separation of a cell homogenate.

Solubility of ProteinsSalting in: When proteins are placed in an aqueous solution, the only ionic species in solution are the other protein molecules. Water, although polar, is only slightly ionized so the proteins tend to aggregate based on ionic interactions that form between themselves. The interactions between protein molecules are more favorable than interactions between water and a protein.

At low salt concentration (NaCl), other ionic species are now present to compete with the ionic protein:protein interactions. As a result, the ionic interactions between proteins break up and the proteins dissolve. Both the small ions (from NaCl) and the proteins are solvated by water.

Solubility of ProteinsSalting out: At high salt concentration (typically with (NH4)2SO4 or Na2SO4), water molecules are more strongly attracted to these small ions (especially multivalent ions) than to the large protein molecules. The proteins are left then to seek whatever favorable interactions exist and these are the protein:protein associations which result in aggregation and precipitation.

Isoelectric precipitation: At the pI there is zero net charge on a protein. At a pH away from the pI, each protein molecule bears an identical charge (either + or - depending on the pH) resulting in repulsion between molecules. At the pI, no repulsion occurs, and the proteins will aggregate and precipitate.

Determining the isoelectric point (pI)

Isoelectric point: The pI is the pH at which there is zero net charge on a molecule. Look at Asp.

The zero net charge form is a part of the first two ionizations. Therefore, the maximum amount of this is present at a pH of (2.09 + 3.86)/2 = 2.98 = pI.

HOOC-CH2-CH-COOH

NH3+

HOOC-CH2-CH-COO-NH3

+

+ H+ 2.09

HOOC-CH2-CH-COO-NH3

+

-OOC-CH2-CH-COO-NH3

+

-OOC-CH2-CH-COO-NH3

+

-OOC-CH2-CH-COO-NH2

+ H+ 3.86

+ H+ 9.82

Dialysis

Separation ofvery large from very small molecules is based on anattempt to equilibrateconcentration.

Osmotic pressure

Gel Filtration – size separation

Affinity Chromatography

HPLC(up to 5000 psi)

Proteins can be detected from absorbance of the peptide bonds in the uv at 220 nm.

However, it is morecommonly doneat 280 nm as seenearlier.

Ion Exchange Chromatography

Depends upon the charge on each of the various molecules being separated.

Ion Exchange Resins

Determining the Charge on a Peptide

Determing the charge on a peptide involves a knowledge of ionic equilibria, pKas and the ionic forms present at a given pH.

In a peptide, the amino terminus, the carboxy terminus and the ionizable side chains may be charged at a given pH.

The sum of these charges gives the net peptide charge.

Electrophoresis

Separation of molecules by electrophoresis depends upon:

1. the strength of the electric field (voltage), 2. the charge on each molecule,3. the frictional coefficient of movement

through the solid support which in turn depends on the radius or mass of the molecule.

So, essentially electrophoresis separates based on a charge/mass ratio.

Electrophoresis

A classical electrophoresis apparatus.

V

buffer buffer

solid support for sample

cooling plate

Sodium Dodecyl Sulfate

SDS is an anionic detergent that binds uniformly along a protein chain. About one SDS binds for every two amino acid residues. Thus all proteins bear the same charge/mass ratio and separation by electrophoresis will be based on mass alone.

SDS Gel Electrophoresis

Proteins after Staining

Isoelectric Focusing

Electrophoresis on a mixture of polyampholytes.

Each molecule migrates to its point of zero net charge.

SDS PAGE after IEF

Sequential Purification Steps

SDS electrophoresis

UltracentrifugationThe S value is a measure of the rate of sedimentation,(a sedimentation coefficient) and is not linear with MWbecause of molecular shape.

In a sucrose or cesium chloride gradienta molecule migrates thebuoyant density equal toits own.

Amino Acid AnalysisDetermines amino acid composition of a protein.A protein is hydrolyzed in 6N HCl, 24 hrs at 100oC.Separation of AA by ion exchange chromatography.

Detecting Amino Acids

Classical reagent for amino acids. Reaction requires 2-5 min at 100oC and givesnanomole level detection.

Ruhemann’s Purple570 nm

OH

O

O

OHNH2-CH-COOH

CH3

+

O

O

O

O

NCO2CH3

CHO+ +2

Detecting Amino Acids

Reacts immediately with primary amines at roomtemperature. Detection at picomol level due to fluorescent products

Automated Sequencing

Edman’s Method

Edman degradation procedure - Determining one residue at a time from the N-terminus

(1) Treat peptide with phenyisothiocyanate (PITC) at pH 9.0 which reacts with the N-terminus to form a phenythiocarbonyl (PTC)-peptide.

(2) Treat the PTC-peptide with anh. trifluoroacetic acid (TFA) to selectively cleave the N-terminal peptide bond and form a triazolinone derivative.

(3) Extract N-terminal derivative from the peptide.

(4) Rearrange to a phenylthiohydantoin (PTH)-amino acid with aq. HCl then chromatograph.

Edman’sMethod

Phenylthio-hydantoin(PTH)

PITC

Edman’s Method

N=C=S

phenylthiocarbamyl-peptide

+ NH2 - CH - C - N - CH -C

O

H O

CH2-OH

CH3

NH - C -

S

NH - CH - C - N - CH -C

O

H O

CH2-OH

CH3

S

N

O

CH3NH -+

N

N

O

CH3S

NH2 - CH - C

O

CH2-OH

thiazolinone derivativephenylthiohydantoin derivative

Separation of PTH-AAs by HPLC

N-Terminal Reagents

DNFB - Sanger’s reagent (dinitrofluorobenzene)

DANSYL choride (dimethylaminonaphthalenesulfonyl chloride)

NO2

NO2

F

DNFB

SO2Cl

N(CH3)2

DANSYL-Cl

Other ReagentsC-terminal:

Hydrazine

Disulfide reduction:Dithiothreitol - Cleland’s Reagent

Thiols:Iodoacetate

5,5’-dithiobis-(2-nitrobenzoic acid) - Ellman’s reagent

NH2-NH2

HO C H

H C OH

CH2 -SH

CH2 -SH

Cleland's

NO2 S S

COOH

NO2

COOHEllman's

I-CH2-COOH

Protein Cleavage

Protein sequencing is most manageable with small polypeptides.

Therefore, in order to sequence a large protein, it must be cleaved into smaller pieces.

Cleavage is conducted using either chemical or enzymatic methods.

The pieces must be separated and purified before sequencing.

Chemical and Enzymatic Cleavage

CNBr Cleavage at Met

CNBr Cleavage

at Met

Enzymatic cleavage by Trypsin

An Example, Peptide overlap

Dithiothreitol Reduction of –S-S-

Iodoacetate reaction with -SH

Performic acid oxidation of –S-S-

Sequencing using DNA

Edman’s degradation has been a tremendous asset in protein sequencing, however, for larger proteins recombinant DNA technology is now being used.

Merrifield Solid-Phase SynthesisMerrifield and coworkers at Rockefeller Institute devised a revolutionary solid phase method of protein synthesis in 1963. The proved that this method was effective for larger proteins by synthesizing ribonuclease, an enzyme with 124 amino acid residues.

A chloromethylated polystyrene polymer (resin) was used as the solid support.

Dicyclohexylcarbodiimide was used as an amino acid activator and a water scavenger in the condensation reaction.

Merrifield received a Nobel Prize in 1984 for this work.

Other Methodologies

Immunochemistry: (omit as this is in section 3.3)ELISA = Enzyme-linked immunosorbent assay

Western blottingFluorescense microscopy

Mass Spectrometry: (section 3.5)MALDI = matrix-assisted laser desorption- ionization TOF = time of flight

X-ray Crystallography: (section 3.6)

Nuclear Magnetic Resonance: (section 3.6) NOESY = nuclear Overhauser enhancement spectroscopy.

• Amino acid sequence data provide a basis for preparing antibodies specific for

a protein of interest.

• Amino acid sequence are valuable for making DNA probes that are specific for

the genes encoding the corresponding proteins.

• The nucleotide sequence of DNA (gene) directly reveals the entire amino acid

sequence of the protein encoded by the gene.

• However, DNA sequence can not disclose the information regarding post-

translational modification.

Practical Usage of Amino Acid and DNA Sequences

Antibody

• Antibody (immunoglobulin) is a protein synthesized by an animal in response to

the presence of a foreign substance (antigen).

• Antibodies have specific and high affinity against antigens.

• Proteins, polysaccharides and nucleic acids can be effective antigens.

• Epitope : a specific group or cluster (portion) of antigen to stimulate the

synthesis of an antibody and recognized by a specific antibody (antigenic

determinant)

• Hapten : a small molecule containing epitope attached to a carrier

Antibody (continued)

• Each antibody producing cell synthesizes only one type of

antibody recognizing a single kind of epitope.

• The proliferation of a given antibody producing cell is

stimulated by the binding of its designated antigen to the

cell surface receptor of the antibody producing cell .

• Periodic injections of an antigen into the host animal can

raise the antibodies specifically recognizing the injected

foreign substance.

• Blood withdrawn from the immunized host animal

centrifugation separation of blood cells (pellet) and

serum (supernatant) anti-serum

• Anti-serum contains multiple kinds of antibodies each

recognizing a different surface feature of the same

antigen.

• This heterogenic antibodies are called as polyclonal

antibodies.

• This heterogeneity can complicate the use of these

antibodies.

Monoclonal Antibody

• Monoclonal hybridoma cell lines can generate large amount of homogeneous antibodies.

• Monoclonal antibodies can serve as precise analytical, preparative and therapeutic reagents.(HCV, HIV, herceptin) Immuno-

Staining ofDrosophila

Embryousing

MonoclonalAntibodyagainst

Engrailed

Plasma cell

by antigen-antibody interaction

Monoclonal antibody drugs?

Herceptin Binds to the C-terminus of Domain IV

Herceptin Fab

I

III

II

IV

N

C

HER2

Ribbon Diagram of Her-3 ECD

N

C

“Tethered”I

II

III

IV

Right-handed β helixLaminin-like folds

Fig. 14.28 pp397

Surface representations of EGFR and HER2 in Antibody-Bound Conformations

Herceptin

ELISA (Enzyme-Linked Immuno-Sorbent Assay)

Antibody detection, anti-HIV antibody

Antigen detection

Western Blotting

Radioactive secondary antibody

For protein expression and purification

Immuno-FluorescenceMicroscopy

Actin Filament Stainingusing α-actin antibody

Immuno-ElectronMicroscopy

Detection of a channel proteinfrom the synaptic vesicles

using antibodies tagged withelectron-dense markers such as gold

or ferritin(Resolution better than 10 nm)

Fluorescence-labeled antibodies(resolution 200nm)

ex) Glucocorticoid receptor

Time of Flight Mass Spectrometer

Nuclear Magnetic Resonance

End of Chapter 3

Thank You