Figure 2. Activity and size of HEWL aggregates.

a, Bacteriolytic activity (in ▵Abs/min) of HEWL (diluted to 70 nM in assay cuvette) measured at pH 7 with pppppppwith M. lysodeikticus is highlighted at different time intervals subsequent to incubation of HEWL at concentrations 0.7, 3, 50 & 120 µM in pH 12.2 and 120 µM in pH 7. b, Changes in steady state fluorescence anisotropy (r_ss) of dansyl-HEWL conjugates at different time intervals in presence of 0.3, 3, 20, 50 & 120 µM of HEWL incubated in pH 12.2 and 50 µM in pH 7 is shown along with fitted curve for pH 12.2 (Eq. 2, Table 1). c, Nanosecond time-resolved fluorescence anisotropy decay of dansyl-HEWL conjugates after 24 hours for following protein concentrations (pH): A: 3 µM (7); B—E: 0.3, 3, 20 and 120 µM (12.2). The dark continuous line depicts the fit and extracted value for φ₂ using tail fit analysis (Table S1). d, Average global rotational time (φ₂) for 0.3—120 µM HEWL at pH 12.2 and 3 µM at pH 7 (extreme left) after 12 & 24 hours (Table S2). e, Size of HEWL aggregates revealed by dynamic light scattering after incubation of 120 µM HEWL in pH 12.2 (continuous line) and pH 7 (dashed line) for 26 hours. Peaks at R_h ∼2.0 nm indicate HEWL monomer, while those at 12.5 and 17.4 nm along with shoulders at R_h ∼9.0 and 28.7 nm reveal multimeric aggregates. Error bars, SD; N  =  3.