EPR CHARACTERIZATIONS OF ALPHA-CHYMOTRYPSIN ACTIVE-SITE DYNAMICS IN REVERSED MICELLES AT ENHANCED GAS-PRESSURES AND AFTER SUBJECTION TO CLATHRATE FORMATION CONDITIONS

Electron paramagnetic resonance spectroscopy is used to characterize t he active site dynamics of alpha-chymotrypsin solubilized in reversed micelles. Of particular interest is the behavior of the enzyme when th e micellar system is subjected to enhanced gas pressures and low tempe ratures. At specific thermodynamic conditions, clathrate hydrates form from the intramicellar water, reducing the micelle size and water con tent. Also, beyond a critical pressure, micellar instability results. The EPR spectra under these conditions indicate that the rotational co rrelation times increase appreciably only when the water-to-surfactant molar ratio, w0, is reduced to values lower than 10. The EPR characte rization also reveals a remarkable resilience of the enzyme when subje cted to pressure-induced changes; when returned to ambient conditions, activity and active site dynamics are fully restored. (C) 1994 John W iley & Sons, Inc.