Glutaraldehyde In Buffer Research Articles

Effects of stabilizing additives on the activity of alpha-chymotrypsin in organic solvent

Abstract The ability of ectoin, hydroxyectoin, glycoin (known as extremolytes), glutaraldehyde (GA) and methoxypoly(ethylene glycol) (MeOPEG) to produce formulations of alpha-chymotrypsin (α-CT) more active than the native enzyme in organic solvents, was tested. The activity in toluene of lyophilized α-CT when ectoin, MeOPEG (1:1, w/w) or GA (0.1%, w/v) were used was 10-, 46-, and 260-fold higher than in the absence of additives, respectively; furthermore, when using MeOPEG and GA together the activity increased 370-fold. GA increased the activity of α-CT in organic solvent, in spite of its inactivating effect in water. The activating effect of GA was dependent on the water activity ( a w ) at which α-CT + GA was equilibrated and on the reaction system. At a w 0.06 it was observed at all GA concentrations employed (0.025–0.25%, w/v, in the enzyme water solution before lyophilization), whereas at a w 0.96 it was found only at 0.025% GA. Infrared spectroscopic data indicated that prolonged exposure of α-CT films to GA in buffer causes changes to the enzyme secondary structure, favoring the hydration of the enzyme, which ultimately suggests a higher accessibility of the substrates to the enzyme.

Three-dimensional analysis of vacuoles and surface invaginations of capillary endothelia in the eel rete mirabile

One layer of attenuated endothelia of continuous capillaries provides a partially selective diffusion barrier between the blood and the interstitium. Ultrastructures of membrane specialization without the known physiologic functions have been found in blood vessel endothelia. The vacuolar profiles or vacuole-like, membrane-bound structures, which are larger than plasmalemmal vesicles, have been observed routinely in normal endothelial cytoplasm or in blood vessels challenged by insults in electron microscopic studies. Three-dimensional information from serial sections is required to understand the organization and functions of vacuole-like structures in capillary endothelium. The capillaries in eel retia mirabile were perfused with electron-dense tracers, glutaraldehyde in buffer, and were processed for transmission electron microscopy. Ribbons of serial thin sections without counterstaining were examined under a transmission electron microscope. The vacuolar profiles inside endothelial cytoplasm were investigated with the techniques of serial section analysis and three-dimensional reconstruction from serial sections. The vacuole-like structures inside endothelial cytoplasm either were connected to extracellular (luminal, abluminal) compartments or existed as isolated vacuoles from serial section analysis. In the eight series examined in this study, six of ten vacuole-like structures were classified as isolated vacuoles inside endothelia, and their diameters ranged between 186 nm and 266 nm. Two of ten vacuole-like structures were found to extend to the luminal surface of capillaries as luminal, pocket-like invaginations. One of ten vacuole-like structures was found to be connected to the albuminal compartment, and another one existed as an extracellular compartment surrounded by endothelia. Three-dimensional projection of the vacuolar compartments from serial sections showed that endothelial cytoplasm of sheet shape protruded and folded over adjacent endothelium. Three-dimensional information from serial sections reveals the organization of vacuolar profiles and pocket-like invaginations from the cell surfaces in capillary endothelium. The vacuolar profiles in capillary endothelia in two-dimensional electron photomicrographs may represent the extracellular compartments surrounded by the endothelial finger-like extensions. The results indicate that the luminal and abluminal surfaces of the capillary lumen are not smooth or static, and endothelia may change their shape in three dimensions through cytoplasmic protrusions when the tissue environment changes.