THIOL DISULFIDE EXCHANGE BETWEEN SMALL HEAT-SHOCK-PROTEIN-25 AND GLUTATHIONE/

Murine small heat shock protein 25 (Hsp25) carries a single Cys-residu e at position 141 of its amino acid sequence. In glutathione redox buf fers, Hsp25 equilibrates between reduced protein (PSH), mixed disulfid e (PSSG) and protein dimer (PSSP) forms. At highly oxidative condition s, native Hsp25 predominantly forms PSSP while denatured Hsp25 forms P SSG. Conversion of PSSP to PSSG correlates with urea and temperature d enaturation of tertiary and/or quaternary structure of Hsp25. At pH 7. 5, 25 degrees C, the second-order rate constant for the formation of P SSP in the reaction of native PSH with GSSG is 20.1 +/- 1.4 M-1 min(-1 ). This is approximately 3-fold lower than the reaction velocity of GS SG with a typical, unhindered thiol of pk(a) 8.6. At redox equilibrium , the fractions of PSSP, PSSG, and PSH depend on the concentration of GSH and less on the ratio [GSH]/[GSSG] (R). At a constant R, the fract ions of PSSG and PSH species depend similarly on GSH concentration, Be ing approximately equal in glutathione redox buffers with low R. It is concluded that in oligomeric complexes, Hsp25 subunits in vitro form stable dimers, in which the reacting -SH groups are in a proximity to form intersubunit disulfide bonds. Within a reaction of one of these - SH groups with GSSG, steric hindrances and electrostatic repulsion com plicate penetration of another reduced or oxidized glutathione molecul e to the reaction site. (C) 1998 Elsevier Science B.V. All rights rese rved.