6533b7d5fe1ef96bd126506c

RESEARCH PRODUCT

Keyhole limpet haemocyanin: negative staining in the presence of trehalose

subject

description

Abstract Samples of unpurified and purified haemocyanin from the giant keyhole limpet Megathura crenulata have been studied by transmission electron microscopy (TEM) using mixtures of trehalose with the negative stains, uranyl acetate and ammonium molybdate. Trehalose is a known protein preservative during air and freeze drying, UV irradiation and high temperatures, and therefore offers the possibility of protecting proteins during the drying of negatively-stained specimens and their subsequent electron microscopical study. Evidence is presented that trehalose possesses satisfactory stability within the electron beam during conventional room temperature, negative-staining studies. The combination of negative stain and trehalose generates a deeper layer of amorphous stain around and within the keyhole limpet haemocyanin (KLH) di- and multidecamers, thereby increasing protein mobility and the generation of a range of tilted images, alongside the more usually observed end-on and side-on image projections. The concentration of uranyl acetate and ammonium molybdate has been increased to 4 and 5%, respectively, in the presence of 1% trehalose, in order to generate satisfactory image contrast. This is because of the reduction by trehalose of the net mass thickness of the dried negative stain-carbohydrate mixture compared to for an equivalent thickness of stain alone. The study of these specimens at low temperatures (−175°C) offers the possibility of superior structural preservation.