SUPRAMOLECULAR ASSEMBLY BETWEEN NANOPARTICLES OF HYDROPHOBIZED POLYSACCHARIDE AND SOLUBLE-PROTEIN COMPLEXATION BETWEEN THE SELF-AGGREGATE OF CHOLESTEROL-BEARING PULLULAN AND ALPHA-CHYMOTRYPSIN

Complexation between alpha-chymotrypsin (Chy) and the self-aggregate o f cholesterol-bearing pullulan (CHP) was studied by size exclusion col umn chromatography (SEC), fluorescence spectroscopy, circular dichrois m (CD), and differential scanning calorimetry(DSC). The CHP self-aggre gate strongly complexed with the Chy dimer and formed colloidally stab le 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 m ay locate deeply inside the matrix of the CHP self-aggregate hydrogel. Upon complexation, the bulk structure of the CHP aggregate changed, t he helix content of Chy increased (from 9 to 29%), and the beta-form c ontent 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 add ition of bovine serum albumin (BSA). Released Chy had almost the same enzymatic activity as that of free Chy before the complexation. The se condary 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 stil l had 74% of the original enzyme activity. No thermal unfolding of Chy in the complex was suggested by DSC and fluorescence spectroscopy ove r 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.