Fig. 2.

The reversible effect of lyophilization. (A) ¹H NMR spectra of 2 mM α-lytic protease in 0.05 M KCl at 600 MHz, 5°C, and pH 4.0 showing the reversible effects of lyophilization and pressure upon the low-field ¹H. Each spectrum represents 1,024 scans using the watergate water-suppression sequence. Spectra shown are for: freshly prepared sample (a), after lyophilization (b), after 60-psi pressure treatment (c), and after a second lyophilization (d). (B) ¹H NMR spectra of 2 mM α-lytic protease in 0.05 M KCl at 500 MHz, 5°C, and pH 8.5 showing the broadening of the high-pH species after lyophilization. Each spectrum represents 512 scans using the hard 1-1 water suppression sequence. Spectra shown are for: freshly prepared sample (a) and after lyophilization (b). (C) Effect on specific activity. Each bar represents the percentage of specific activity of the treatment group compared with untreated enzyme (control) on the substrate Ac-Ala-Pro-Ala-p-nitroanilide (14). Data are shown for: NONLYO with no treatment assayed at the same time points as the other samples (a), freshly lyophilized enzyme resuspended in water (b), lyophilized enzyme subsequently treated with 60 psi of N₂ gas pressure overnight (c), resuspended lyophilized enzyme subsequently relyophilized (d), and twice-lyophilized enzyme that was treated with 60 psi of pressure overnight (e). Each bar represents an average of six experiments. Pressure had no effect on NONLYO (data not shown).