The Effect of Compactional Pressure on a Wheat Germ Lipase Preparation

Abstract

Wheat germ lipase is a relatively homogeneous proteinaceous enzyme known to suffer inactivation by compactional pressure. However, earlier investigators suggested that the inactivation was due to thermal degradation following the application of mechanical energy. A wheat germ preparation (Sigma) was compacted over a range of pressures from 85 to 1800 MPa. The 100-mg compacts were carefully dispersed and dissolved in aqueous 0.05 M potassium phosphate buffer, pH 7.4, and the biological activity was determined using triacetin as a substrate. No significant loss of activity occurred up to a pressure of 175 MPa. After this point, a discontinuity was evident with a loss of 30% activity but this loss of activity remained constant over the remainder of the applied pressure range studied. The density/applied pressure relationship indicated that the discontinuity was due to space constraints. The density failed to go higher than a limiting value of approximately 1.2 g cm⁻³, irrespective of the applied pressure. There was an approximately linear relationship between the relative loss of biological activity and density, indicating that the observed loss of biological activity is unlikely to be due to applied thermal energy but more likely to be due to space constraints on the volume occupied by the molecule. However, circular dichroism measurements and SDS-PAGE examination did not reveal any obvious protein structural changes, suggesting that the mechanism involved in activity loss is subtle.