COUNTERACTING EFFECTS OF THIOCYANATE AND SUCROSE ON CHYMOTRYPSINOGEN SECONDARY STRUCTURE AND AGGREGATION DURING FREEZING, DRYING, AND REHYDRATION

Studies of numerous proteins with infrared spectroscopy have documente d that unfolding is a general response of unprotected proteins to free ze-drying, Some proteins that are unfolded in the dried solid aggregat e during rehydration, whereas others refold, It has been proposed for the latter case that aggregation is avoided because refolding kinetica lly outcompetes intermolecular interactions. in contrast, with protein s that normally aggregate after rehydration, minimizing unfolding duri ng freeze-drying with a stabilizer has been shown to be needed to favo r the recovery of native protein molecules after rehydration. The purp ose of the current study was to examine first the opposite situation, in which a denaturant is used to foster additional unfolding in the pr otein population during freeze-drying, Ii the protein is not intrinsic ally resistant to aggregation under the study conditions (e.g., becaus e of intermolecular charge repulsion) and the denaturant does not disr upt intermolecular interactions during rehydration, this treatment sho uld favor aggregation upon rehydration. With infrared spectroscopy we found that at concentrations of the denaturant Na thiocyanate (NaSCN) that only slightly perturbed chymotrypsinogen secondary structure in s olution before freeze-drying, there was a large increase in protein un folding in the dried solid and in protein aggregation measured after r ehydration, Bands assigned to intermolecular beta sheet were present i n the spectra of samples dried with NaSCN, indicating that aggregation could also arise in the dried solid. By examining the protein structu re in the frozen state, we determined that in the absence of NaSCN the protein remains native. NaSCN caused structural perturbations during freezing, without the formation of intermolecular beta sheet, that wer e intermediate to structural changes noted after freeze-drying. In con trast, samples treated in the presence of NaSCN and sucrose had native -like spectra in the frozen and dried states, and much reduced aggrega tion after rehydration, These results indicate that during freezing an d drying the sugar can counteract and mostly reverse the structural pe rturbations induced by NaSCN before and during these treatments.