KEYHOLE LIMPET HEMOCYANIN - NEGATIVE STAINING IN THE PRESENCE OF TREHALOSE

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 p reservative during air and freeze drying, UV irradiation and high temp eratures, 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 po ssesses satisfactory stability within the electron beam during convent ional room temperature, negative-staining studies. The combination of negative stain and trehalose generates a deeper layer of amorphous sta in around and within the keyhole limpet haemocyanin (KLH) di- and mult idecamers, thereby increasing protein mobility and the generation of a range of tilted images, alongside the more usually observed end-on an d side-on image projections. The concentration of uranyl acetate and a mmonium molybdate has been increased to 4 and 5%, respectively, in the presence of 1% trehalose, in order to generate satisfactory image con trast. This is because of the reduction by trehalose of the net mass t hickness of the dried negative stain-carbohydrate mixture compared to for an equivalent. thickness of stain alone. The study of these specim ens at low temperatures (-175 degrees C) offers the possibility of sup erior structural preservation.