Properties Size - change covalent structure - change folding - aggregation state Solubility - change charge (pH or pI) - dielectric constant of solvent - ionic strength of solvent Charge - change pH or pI pI - chemical or enzymatic modification Hydrophobicity - chemical or enzymatic modification Function - depends on the function...

Treatments pH Dielectric constant Salt concentration Hydrolysis Temperature Chemical modification Enzymatic modification

pH Proteins have many acidic groups (-COOH) which are negatively charged at neutral pH and many basic groups (LysNH 2, Arg-guanidino) groups which are positively charged at neutral pH. These are generally surface groups. At some pH, the isoelectric point or pI, negative and positive charges balance, and the protein has a net charge of zero. Most globular proteins have pI<6.5, and are polyanions at neutral pH. Proteins with a net charge generally tend to repel each other and allow their charged groups to interact with water. Proteins are least soluble at their pI, and proteinaceous structures (e.g., muscle fibers) tend to compact.

Dielectric Constant Availability of solvent water and the ability of water to decrease intermolecular attraction keeps globular proteins in solution. In a vacuum:, in a medium Water has a dielectric constant of ~80 and is relatively good at keeping opposite charges apart. Dielectric constants of water-miscible solvents: Glycerol42.5 Ethanol24.3 Acetone20.7 Isopropanol18.3 Mixing one of these solvents with aqueous solutions can decrease the solubility of proteins

Salt Concentration Salts compete for water of solvation At high concentrations, the salts bind the water that was necessary to solvate proteins, and the proteins seek other interactions. If they associate with one another, they precipitate. Solubility depends on 1) the protein 2) the salt 3) the ionic strength, I

Salt Concentration Idealized case (compare with previous slide) If K’S values are different then salting-out fractionation may be possible

Salt Properties Lyotropes. Salts that favor water structure, and strengthen the hydrophobic effect Chaotropes. Salts that disrupt solvent structure, weakening the hydrophobic effect (solubilizing membrane proteins, weakening globular protein folds)

Hydrolysis 6N HCl, 110°, reduced pressure, 20 hr free amino acids, Trp destroyed No longer proteins Enzymic (endoprotease) limited - mixtures of peptides exhaustive - limit peptides (mixture) Often greater solubility, generally lower water-holding capacity, lower foamability, probably lower emulsification Increased digestibility (duh)

Temperature Most prominent effect - unfolding Increase in partial specific volume Increase in shape parameter Increase in [  ] and observed viscosity Aggregation Depending on T, rate of heating, I, etc., can get Gel (loose network entrapping much solvent) Cooked precipitate Melted cheese (!)

Chemical Modification Succinic anhydride - converts Lys-NH 3 + to R-COO - alters pI and charge at fixed pH creates net anionic surface (functionality) Acetic anhydride- converts Lys-NH 3 + to R-COO - alters pI and charge at fixed pH ; retards Maillard browning Aminoacyl anhydrides-add more amino acids Carbodiimide/amines or carboxylic acids Reductive methylation-aldehyde or ketone followed by borohydride; retards Maillard browning Crosslinking - glutaraldehyde, dimethyl suberimidate Result depends on the reagent

Enzymatic Modification Hydrolysis - discussed already Dephosphorylation, deglycosylation (hard) Transpeptidation - the Plastein Reaction* Use enzymatic hydrolyzate, concentrate Add protease plus free amino acids or esters Obtain small, reshuffled protein-lets (3 kD) with new amino acids incorporated Actual composition depends on the mix and the specificity of the protease used. Transesterification* -glycosidases can reshuffle sugars Lactase is specific for beta-galactosides invertase is specific for beta-fructosides Neither cares very much what the other reagent is. *(catalysts catalyze both forward and reverse reactions)