Transcript Catalysis - University of California, Davis

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
(LysNH2, 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.
z iz j
zizj
In a vacuum: force  2 , in a medium force 
Dri,2j
ri, j
Water has a dielectric constant of ~80 and is relatively good
at keeping opposite charges apart. Dielectric constants of
water-miscible solvents:
Glycerol
42.5
Ethanol
24.3
Acetone
20.7
Isopropanol
18.3
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
mi zi
I
2
i
Salt Concentration
slope = K's
(~1/2 m)
Idealized case
(compare with previous slide)
I
If K’S values are different
then salting-out fractionation
may be possible
log (solubility)
log (solubility)

Enzyme 3
Enzyme 1
Enzyme 2
20%
40%
60%
Ammonium sulfate (% of saturation)
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
Result depends on the reagent
•Succinic anhydride - converts Lys-NH3+ to R-COO•alters pI and charge at fixed pH
•creates net anionic surface (functionality)
•Acetic anhydride- converts Lys-NH3+ 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
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)