Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase

During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native protein in the dried state as well as the complete recovery of active beta-gal on reconstitution. These results indicate that addition of an effective stabilizer, such as sucrose, may minimize protein denaturation during freeze-drying in phosphate buffers, even if there are large-scale changes in solution pH during freezing.