Przybycien, Todd Michael (1989) Structure, function and aggregation kinetics in salt-induced protein precipitation. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-02202007-153541
Salt-induced precipitation is a biological separation technique that exposes proteins to unnatural environments. Macromolecular-scale issues of activity, structure, and aggregation have been addressed as a function of governing parameters.
The effects of salt type and concentration on protein solubility and recoverable activity were studied using [alpha]-chymotrypsin ([alpha]CT) as a model protein and five salts spanning the lyotropic series. Unaccounted for salt-protein interactions and changes in protein physical properties were the likely source of discrepencies between the experimental and theoretical solubility behavior. Active protein recovery was a function of salt type, but not concentration. A salting-out performance parameter was identified; an optimum salt may exist for a particular protein.
[alpha]CT precipitates from the solubility-activity study were examined for perturbations in secondary structure via Raman spectroscopy and in active site tertiary structure via electron paramagnetic resonance spectroscopy. NaBr, KBr, and KSCN-induced precipitates had increased [beta]-sheet and decreased [alpha]-helix contents; these changes were correlated with active protein yields. Spectra of spin-labelled precipitates indicated that the active site remains intact. Molecular modelling was used to estimate changes in the dipole moment and hydrophobic surface area for the altered precipitates. A general mechanism for the precipitation of globular proteins was proposed.
The generality of secondary structure changes was explored for twelve different proteins via Raman spectroscopy. KSCN-induced precipitates exhibited increased [beta]-sheet and decreased [alpha]-helix contents; structural changes for NaSO-induced precipitates were less significant. The [beta]-sheet increase may occur at the expense of [alpha]-helix segments. [beta]-sheet increases were correlated with the fraction of charged residues and the surface area of the native protein. [alpha]-helix decreases were correlated with the dipole moment and helical content of the native protein.
The effects of temperature, protein concentration, salt type, and salt concentration on [alpha]CT aggregation kinetics were studied. Stopped-flow turbidimetry indicated that temperature and salt concentration effects are exerted through changes in protein solubility. Protein concentration effects are well-described by Smoluchowski's collision equation. The aggregation of partially inhibited [alpha]CT demonstrated poisoning behavior. Solute particle radius distributions determined by dynamic laser light scattering indicated that aggregation denends on the supersaturation. A detailed population balance model, accounting for specific and nonspecific quaternary interactions, was developed
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||10 March 1989|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||14 Mar 2007|
|Last Modified:||26 Dec 2012 02:31|
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