Structure, Function and Aggregation Kinetics in Salt-Induced Protein Precipitation

Citation

Przybycien, Todd Michael (1989) Structure, Function and Aggregation Kinetics in Salt-Induced Protein Precipitation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/2atq-2y42. https://resolver.caltech.edu/CaltechETD:etd-02202007-153541

Abstract

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 α-chymotrypsin (αCT) 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.

αCT 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 β-sheet and decreased α-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 β-sheet and decreased α-helix contents; structural changes for Na₂SO₄-induced precipitates were less significant. The β-sheet increase may occur at the expense of α-helix segments. β-sheet increases were correlated with the fraction of charged residues and the surface area of the native protein. α-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 αCT 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 αCT 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.

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