Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were used to investigate the thermal conformational change associated with the activity loss of spinach Rubisco. CD and intrinsic fluorescence demonstrated a three stage thermal unfolding of Rubisco. At 25-45degreesC, the secondary structure did not change but the tertiary and/or quaternary structure changed obviously with increased temperature. In 45-60degreesC, the secondary structure showed much change with increased temperature and the tertiary and/or quaternary structure changed much faster. Over 60degreesC, whole conformation changed abruptly with increased temperature and finally unfolded completely. DSC, CD and activity assays after annealing showed that the conformational change and the activity loss of Rubisco were completely reversible if the heating temperature was below 45degreesC, partly reversible between 45 and 60degreesC, and irreversible beyond 60degreesC. (C) 2002 Elsevier Science (USA).