COLD-INDUCED CONFORMATIONAL-CHANGES OF RIBONUCLEASE-A AS INVESTIGATEDBY SUBZERO TRANSVERSE TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

Subzero temperature gradient gel electrophoresis is a new approach whi ch allows to measure the transition temperature of low temperature-ind uced subtle conformational changes of proteins and to detect the diffe rent conformational states, including unfolded states. Using this tech nique under destabilizing conditions, i.e., in the presence of 4 M ure a, bovine pancreas ribonuclease A exhibited two transitions: (i) a con tinuous transition with a midpoint temperature of - 14 degrees C corre sponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discon tinuous transition at - 22.5 degrees C from intermediate to a non migr ating species. Under reducing conditions this second transition was sh ifted toward high temperatures(-18.5 degrees C). We attempted to detec t these two transitions by differential scanning calorimetry, UV spect rophotometry and circular dichroism measurements. These transitions ha ve been ascribed to subtle cold-induced conformational changes.