Fig. 3.

Macromolecular crowding enhances the initial rate and the overall extent of consumption of Trp residues. The initial rate of Trp consumption (Ri_Trp) was determined in the absence or presence of the crowding agent dextran (Mw 35,000–45,000) by following the loss of the intrinsic fluorescence of Trp (λ_ex: 295 nm; λ_em: 360 nm). The length of radical chain reactions was determined from the ratio of the total number of μmoles of Trp consumed per μmoles of ROO• generated after 30 min of incubation in presence of AAPH (100 mM) at 37 °C. Panel A: Kinetic profiles of Trp (50 μM) consumption on incubation with AAPH (100 mM) in phosphate buffer, 30, 60 and 120 mg mL⁻¹ dextran. Panel B: Kinetic profiles of Trp (500 μM) consumption on incubation with AAPH (100 mM) in phosphate buffer, 30, 60 and 120 mg mL⁻¹ dextran. Panel C: Ri_Trp elicited by radicals derived from AAPH (100 mM) in phosphate buffer in the absence (black squares) or presence of 60 mg mL⁻¹ dextran (blue triangles). Panel D: Linear fit of Ri_Trp values as a function of the concentration of the crowding agent dextran at different Trp concentrations. Panel E: Ratio of total Trp consumption (μmoles) per total dose (μmoles) of ROO• generated upon AAPH thermolysis in phosphate buffer, 30, 60 and 120 mg mL⁻¹ dextran. Data are expressed as means ± SD from three independent experiments. Asterisks indicates data significantly different (p < 0.05) to the samples incubated in absence of dextran. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)