The Effect of Certain Fixatives on Particles of a Globular Protein: an Electron-Microscopical Study

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The effect of certain fixatives on particles of a globular protein: an electron-microscopical study

By K. DEUTSCH, E. FISCHER, and W. KRAUSE

(From the Department of Electron Microscopy, University of Greifswald, East Germany)

With one plate (fig. i)

Summary Particles (regarded as molecules) of bovine serum albumin have been studied by electron microscopy both in the untreated state and after fixation with osmium tetroxide, formaldehyde, and potassium permanganate. All three fixatives, especially potassium permanganate, increased the density of the particles considerably.

Introduction A BRIEF report has already been made of a preliminary study of the effect of osmium tetroxide on minute protein particles (Deutsch and others, 1962). The purpose of the work described in the present paper was to extend the study, especially by working with fixatives other than osmium tetroxide and by making measurements of the changes in density caused by fixation.

Materials and methods Specimens of bovine serum albumin were obtained from Sera of Heidelberg and from Professor J. Segal, Department of General Biology, University of Berlin, who himself prepared some of the material used in the investigation. The protein was dissolved in a phosphate/sodium hydroxide buffer solution at pH 7-2, in the proportion of 1 mg of protein to 1 ml of buffer. 100 ml of twice-distilled water was added to 1 ml of the protein solution. For the investigation of the untreated protein a drop of the diluted solution was deposited on a specimen grid coated with a collodion film. The solution was allowed to dry. The following methods were applied to study the effect of fixatives on the particles. (1) A drop of the diluted solution was deposited on the specimen grid, exposed to the vapour of formalin, and allowed to dry in air. (2) Solutions of osmium tetroxide or of potassium permanganate were added to the diluted protein solution, to make the final concentration of the fixative substance 0-2% w/v in the case of osmium tetroxide, or 0-1% w/v in that of potassium permanganate. The solutions were then dialysed to remove all fixative substance that had not attached itself to the protein particles. Small drops of the dialysed solutions were deposited on specimen grids and allowed to dry in air. Collodion filmswer e used throughout, because agglomeration of the protein occurred on carbon films. [Quart. J. micr. Sci., Vol. 105, pt. 2, pp. 227-30, 1964.] 228 Deutsch, Fischer, and Krause Micrographs were taken with a Zeiss D 2 electron microscope working at 48 kV, with instrumental magnification of 18650. A photocell was used as an exposure photometer. The photocell was attached to the screen and con- nected to a sensitive galvanometer (von Ardenne, 1944). The micrographs were enlarged and evaluated densitometrically. Great care was taken to do all electron microscopical and photographic work under the same conditions to enable a quantitative comparison of the staining effect of the various fixatives to be made. For comparison, thin layers of various thicknesses were prepared by evaporating gold on a specimen grid. The mass density of the protein molecules (treated and untreated) was correlated with the thickness of gold films of corresponding mass density. Results and discussion The particles are represented on the electron micrographs as small dots (fig. 1, A to D). The facts are consistent with the belief that each particle represents a protein molecule. The diameter (about 5 m^.) and shape of the particles are in agreement with the results of an electron microscopical investigation by Valentine (i960), who used negative staining for specimen preparation. The danger of artifacts was avoided by examining in the electron microscope blank supporting films and films on which a drop of the diluted buffered solution had been allowed to dry. The instrumental resolution was about 2-0 m/x; hence accurate measurements of the size and shape of the particles were not possible. Working near the limit of resolution of the instru- ment also decreased the accuracy of the densitometric measurements.

TABLE I d (in myx) is defined as the thickness of a gold layer which has the same mass density as a monomolecular layer of protein. untreated about fixed with formaldehyde about z-od fixed with osmium tetroxide about z-od fixed with potassium permanganate about 3mod Inspection of the electron micrographs shows that the particles treated with formaldehyde, osmium tetroxide, or potassium permanganate are much more electron-dense than untreated ones. This is confirmed by the results of the densitometric evaluation (table 1). There are several possible reasons for the increased contrast. (1) It is well known that the mass density of some specimens is reduced in the electron beam, as a result of chemical changes

FIG. I (plate). A, untreated albumin particles. B, albumin particles treated with osmium tetroxide. c, albumin particles treated with formaldehyde. D, albumin particles treated with potassium permanganate. A few of the protein particles (regarded as molecules) have been indicated by arrows. I .*.

FIG. I K. DEUTSCH, E. FISCHER, and W. KRAUSE Effect of fixatives on protein molecules 229 brought about by the effect of the beam on the specimen. It is quite likely that this effect is smaller on a 'fixed' specimen; hence the reduction of the mass density in the beam may be smaller or negligible. (2) It is also likely that treatment with some fixatives increases the mass density considerably. This holds for osmium tetroxide and potassium permanganate, and the possibility cannot be excluded that formaldehyde also reacts with the protein to produce this effect. The particles of protein treated with potassium permanganate have a dark core (diameter about 5 m/i) and are surrounded by a 'halo', presumably a 'deposit' of some sort. This deposit surrounding the molecule also contributes to the mass density. (3) The molecules may contract somewhat as a result of the treatment with fixatives and this effect would also increase the mass density. We have the impression that molecules of protein treated with formaldehyde have a smaller diameter than untreated ones. However this may be, the decisive factor for electron microscopy is the increase of contrast, and it may be said that these fixatives also act as 'electron stains'. It is, of course, tempting to generalize from our results. All globular proteins have a rather similar structure, and the fixatives we have studied probably have a similar effect on many globular proteins, though there will be quantitative differences. The contrast in cells fixed with osmium tetroxide is usually good, and, as our investigations show, this result must be due in part to the effect of this fixative on proteins. In specimens fixed with potassium permanganate, certain structures are very electron-dense. This might indicate that they contain a large amount of protein, as it is conceivable that this fixative has a specific staining effect on proteins. Reference should be made here to a comprehensive study of fixation by potassium permanganate for electron- microscopy, by Bradbury and Meek (i960). These authors found that potassium permanganate is a medium for revealing membrane structures, which are supposed to contain a considerable amount of proteins. This is in agreement with the 'staining' effect that we have observed. But according to Bradbury and Meek, potassium permanganate is not a true fixative: the actual fixation is effected by the treatment with alcohol. In spite of this, potassium permanganate is usually regarded as a fixative, though basic proteins are supposed to be leached out by the action of potassium permanganate and subsequent treatment. These findings show that one has to be very careful in generalizing from the results obtained by studying only one type of protein. Bradbury and Meek also found that a very fine precipitate of manganese dioxide is formed after treatment with potassium permanganate. This deposit is presumably identical with the one found on protein particles treated with potassium permanganate. In specimens fixed by formaldehyde, certain structures are fairly electron- dense. This may be due to a specific 'staining' effect of formaldehyde on proteins; but to decide this question, an investigation would be necessary to find out whether potassium permanganate and formaldehyde 'stain' cellular 230 Deutsch, Fischer, and Krause—Protein molecules constituents other than protein. However this may be, formaldehyde and potassium permanganate do increase contrast by 'staining' protein. It should be pointed out that even untreated protein itself has enough intrinsic contrast to be visible in the electron microscope.

References ARDENNE, M. von, 1944. Kolloid-Z., 108, 195. BRADBURY, S. and MEEK, G. A., i960. Quart. J. micr. Sci., 101, 241. DEUTSCH, K., 1962. Wissenschaftliche Zeitschrift, Ernst-Moritz-Arndt-Universitat Greifs- wald, 11, 71. SEGAL, J., and KALAIDJEV, A., 1963. Nature, 19s, 177. VALENTINE, R. C, i960. Proceedings of the Regional Conference on Electron Microscopy, p. 708.