Analysis of Proteins and Peptides Amino acid composition Molecular weight Isoelectric point Subunit structure Prosthetic groups Solubility Biological activity

Rule: Before a protein can be analyzed, it must first be isolated in a pure state, i.e., purified Rule: Protein purification means separating the protein of interest from other proteins and components Separation Strategies Protein from salt and small ions Protein from other proteins Protein from H 2 O

Protein from H 2 O (Concentration) Salting out (ammonium sulfate) Precipitation Organic solvents (ethanol, acetone) Heavy metals or salts (trichloroacetic acid) Dehydration Lyophilization (cold evaporation) Osmosis Force protein-protein interactions above protein solvent

Protein from salt and small ions Size-dependent diffusion Dialysis Gel filtration Dialysis Start Gel Filtration End

Principle of Gel Filtration Penetrating the beads slows the migration rate of the smaller particles

Protein from Protein Rule: Protein-Protein separations is based on differences in physical properties of the proteins Size or molecular weight Charge at a given pH (isoelectric point) Unique structural features Major: Minor: Solubility in water, salt solutions, or organic solvents Rule: All effective methods for separation of proteins employ the techniques of CHROMATOGRAPHY Rule: All effective methods for separation of proteins employ the techniques of CHROMATOGRAPHY

Chromatography : (lit., separation by color) Mobile phase Stationary phase Types: Column chromatography - gravity or pump driven Electrophoresis - driven by electric current Medium: Porous gels (size exclusion) Charged resins (charge affinity) Gas-liquid (hydrophobic interactions) Affinity resins (group recognition) Moving force Opposing force Paper(no longer in use) Osmotic movement

Properties of Proteins Detecting Proteins Colorimetric Tests Ultraviolet Absorbance at 280 nm Quantifying Proteins Trichloroacetic acid precipitation Bradford analysis Nitrogen content (16%) Kjeldahl test Absorbance at 280 nm E 1% = 10(10 mg/ml) See Strategies pp See Strategies pp 61-67

STRUCTURE ANALYSIS Sequence Analysis Edman Degradation Acid Hydrolysis Composition 6 N HCl, 105 o C, 18 hours Breaks all peptide bonds Sequential removal from N-terminus

CHARACTERIZATION MOLECULAR MASSKilodaltons MOLECULAR WEIGHTNo Units TRUE Molecular Weight RELATIVE Molecular Weight Sedimentation Analysis SDS (Sodium dodecylsulfate) electrophoresis Gel Filtration (M R )

Protein Solution TCA Precipitation White Precipitate Signifies Protein TCA will remove protein from a solution

CHARGE PROPERTIES OH H+H+

Chromatography based on Charge Ion exchange (cation or anion) H + CH 2 -CH 2 -N CH 2 -CH Salt gradient Most negatively charged protein comes off last Diethylaminoethyl (DEAE) group attached to the cellulose DEAE column NaCl eluent

Electrophoresis Movement of a charge particle in an electric field pH-dependent (native protein) Sodium dodecyl-sulfate (SDS) Uniform charge/mass ratio Uses Assess protein purity Determine relative Mwt (SDS) Denatures protein Relative Mwt dependent on chain length

Isoelectric Focusing (IEF) Principal A protein will move in an electric field only when it has a net charge on its structure. Application Establish a stable pH gradient in the medium Mixture of organic acids with different pKa’s (ampholytes) Uses Assess protein purity Determine isoelectric point (pI) of a protein