Thermal unfolding and proteolytic susceptibility of ribonuclease A

With the aim to localize the structural region that becomes first accessible to proteolytic attack during thermal unfolding, the proteolysis of ribonuclease A was studied in the temperature range of 20-65 degrees C. Subtilisin, proteinase K, and elastase proved to be not appropriate as indicators of thermal unfolding, because even the native protein molecule was cleaved by these proteases. In contrast, chymotrypsin, trypsin, and thermolysin attacked ribonuclease A only after its thermal treatment. For thermolysin and trypsin, the first primary cleavage sites of ribonuclease A could be identified by blotting of the electrophoretic bands, partial N-terminal sequencing of the fragments and assignment according to their molecular masses. The results were confirmed by the separation of the proteolytic fragments by HPLC and subsequent matrix-assisted laser desorption ionization mass spectrometry. The first cleavage sites were determined to be Lys31-Ser32 and Arg33-Asn34 for trypsin and Asn34-Leu35 and Thr45-Phe46 for thermolysin. Hence the structural region from Lys31 to Leu35, together with the adjacent beta-structure containing Thr45-Phe46, is suggested to represent a labile region of the ribonuclease A molecule, which becomes exposed at thermal denaturation.