Complexation between alpha-chymotrypsin (Chy) and the self-aggregate of cholesterol-bearing pullulan (CHP) was studied by size exclusion column chromatography (SEC), fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry(DSC). The CHP self-aggregate strongly complexed with the Chy dimer and formed colloidally stable nanoparticles (R(G) = 12 nm) at pH 4.2 and 25 degrees C. Enzymatic degradation of the CHP-Chy complex by pullulanase suggested that Chy may locate deeply inside the matrix of the CHP self-aggregate hydrogel. Upon complexation, the bulk structure of the CHP aggregate changed, the helix content of Chy increased (from 9 to 29%), and the beta-form content decreased (from 34 to 21%). V-max of the complexed Chy at pH 8.0 decreased up to 1/88 compared with that of free Chy at pH 8.0, while K-m did not change much. Chy was released from the complex by the addition of bovine serum albumin (BSA). Released Chy had almost the same enzymatic activity as that of free Chy before the complexation. The secondary structure of Chy in the complex did not change much even after the complex was treated for several hours at 92 degrees C. Even after the heating, Chy was released from the complex by adding BSA and still had 74% of the original enzyme activity. No thermal unfolding of Chy in the complex was suggested by DSC and fluorescence spectroscopy over the temperature range 20-80 degrees C. These results indicated that the thermal stability of Chy dramatically increases upon complexation with the CHP self-aggregate.