Figure 3

(A). Comparison of in vitro chaperone activity of α-crystallin and purified sHsp18. Enzymes (2 U) were heat inactivated (Sma I at 37°C for 90 min and Nde I at 45°C for 90 min) in the presence or absence of molecular chaperones (0.2 μg) and assayed for the cleavage of 1 μg of plasmid DNA. Lanes represent, λHind III marker (lane 1), digested plasmid (lane 2), uncut plasmid (lane 3), plasmid digested with- heat inactivated Sma I (lane 4), heat inactivated Nde I (lane 5), heat inactivated Sma I in the presence of sHsp18 (lane 6), heat inactivated Sma I in the presence of α-crystallin (lane 7), heat inactivated Nde I in the presence of sHsp18 (lane 8) and heat inactivated Nde I in the presence of α-crystallin (lane 9). (B). sHsp18 can act as a molecular chaperone in wide range of physiological temperatures. 0.2 μg sHsp18 or BSA was incubated with 2 U of Sma I at different temperatures for 90 min, and the cleavage of plasmid DNA was assayed at 25°C for 3 hrs. Lanes represent, λHind III marker (lane 1), uncut plasmid (lane 2), plasmid digested with Sma I as control (lane 3), plasmid incubated with heat inactivated Sma I without and with sHsp18 at 30°C (lanes 4–5); at 35°C (lanes 6–7), at 40°C (lanes 8–9), at 45°C (lanes 11–12), plasmid incubated with Sma I with BSA at 35°C, 40°C and 45°C (lanes 12–14). (C). Preheating of molecular chaperones does not affect chaperone activity. Lanes represent, λHind III marker (lane 1), undigested plasmid (lane 2), plasmid digested with- Sma I (lane 3), Sma I with preheated sHsp18 at 100°C for 5 min (lane 4) and with preheated α-crystallin at 100°C for 5 min (lane 5).