Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition 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 that work together as a functional unit.

Protein Concentration from Absorbance Beer’s Law A =  cl The protein absorbance measured at 280 nm is due to Tyr & Trp

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:  mol substrate/min or mol S/sec = katal To follow during purification use Specific activity:  mol 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):  mol substrate/min-  mol E or mol S/sec-mol E = katal/mol E

Centrifugation Separation of a cell homogenate.

Solubility of Proteins Salting 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 Proteins Salting out: At high salt concentration (typically with (NH 4 ) 2 SO 4 or Na 2 SO 4 ), 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 = 2.98 = pI.

Dialysis Separation of very large from very small molecules is based on an attempt to equilibrate concentration. 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 more commonly done at 280 nm as seen earlier.

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.

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

Ultracentrifugation The S value is a measure of the rate of sedimentation, (a sedimentation coefficient) and is not linear with MW because of molecular shape.

In a sucrose or cesium choride gradient a molecule migrates the buoyant density equal to its own.

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

Detecting Amino Acids Classical reagent for amino acids. Reaction requires 2-5 min at 100 o C and gives nanomole level detection. Ruhemann’s Purple 570 nm

Detecting Amino Acids Reacts immediately with primary amines at room temperature. 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’s Method Phenylthio- hydantoin (PTH) PITC

Edman’s Method

Separation of PTH-AAs by HPLC

N-Terminal Reagents DNFB - Sanger’s reagent (dinitrofluorobenzene) DANSYL choride (dimethylaminonaphthalenesulfonyl chloride)

Other Reagents C-terminal: Hydrazine Disulfide reduction: Dithiothreitol - Cleland’s Reagent Thiols: Iodoacetate 5,5’-dithiobis-(2-nitrobenzoic acid) - Ellman’s reagent

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

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 Synthesis Merrifield and coworkers at Rockefeller Institute devised a revolutionary solid phase method of protein synthesis in 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 blotting Fluorescense microscopy Mass Spectrometry: (section 3.5) MALDI = matrix-assisted laser desorption- ionizationTOF = time of flight X-ray Crystallography: (section 3.6) Nuclear Magnetic Resonance: (section 3.6) NOESY = nuclear Overhauser enhancement spectroscopy.

Time of Flight Mass Spectrometer

Nuclear Magnetic Resonance

End of Chapter 3 Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition