Structure-function relationships in the inorganic salt-induced precipitation of alpha-chymotrypsin.
alpha-Chymotrypsin (alpha CT) was used as a model protein to study the effects of salt-induced precipitation on protein conformation. Process parameters investigated included the type and amount of salt used to induce precipitation. The salts studied included Na2SO4, NaCl, NaBr, KBr and KSCN. Precipitate secondary structure content was examined via laser Raman spectroscopy. Conventional and saturation transfer electron paramagnetic resonance spectroscopy were employed to probe the tertiary structure of the active site in spin-labelled alpha CT precipitates. As the molal surface tension increment of the inducing salt increased, the beta-sheet content increased and the alpha-helix content decreased. There was no significant variation in secondary structure with the amount of salt used. The fraction of precipitate that recovered activity on redissolution was correlated with the change in secondary structure content. Spin-labelled precipitate spectra indicated that the active site remains unaltered during precipitation. Molecular modelling was employed to investigate how physical property of alpha CT were affected by these types of conformational change. Estimated physical property changes could not account entirely for observed deviations from current equilibrium theory for salt-induced precipitation. The spectroscopic observations were also combined with activity/solubility results to propose a mechanism for the salt-induced precipitation of globular proteins.
Przybycien
T M
TM
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena.
Bailey
J E
JE
D000818:Animals; D001965:Bromides; D011232:Chemical Precipitation; D002918:Chymotrypsin; D003461:Crystallography; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D011188:Potassium; D017680:Potassium Compounds; D011487:Protein Conformation; D012492:Salts; D012964:Sodium; D012965:Sodium Chloride; D017670:Sodium Compounds; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D013431:Sulfates; D013861:Thiocyanates
D001965:Bromides; D017680:Potassium Compounds; D012492:Salts; D017670:Sodium Compounds; D013431:Sulfates; D013861:Thiocyanates; C012036:sodium sulfate; D012965:Sodium Chloride; D012964:Sodium; D002918:Chymotrypsin; C027938:sodium bromide; C039004:potassium bromide; D011188:Potassium; C009941:potassium thiocyanate