Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification in Paramecium cells.

Abstract

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules. Ultracentrifugation and spectrophotometric analyses revealed the following characteristics of the conjugates: a) They are of a uniform size class; b) their diameters were calculated, and ranged from 5.6 (Fab-H-8-P; H-9-P) to 10.5 (IgG-HRP); c) the persistence of antigen binding capacity was ascertained; d) the deactivation of the marker peroxidase activity due to coupling was as low as 20-30%; e) optimal conditions for use of the electron microscopic (EM) with 3,3'-diaminobenzidine media were elaborated (with a pH optima somewhat different from some standard methods in current use); and f) on the basis of the quantitative data presented, an optimal compromise (either in favor of higher peroxidase activity with HRP conjugates or of smaller size with microperoxidase-Fab conjugates) can be achieved. Finally, the identification of isolated purified actin and of actin in cortical microfilament bundles and ciliary basal bodies of Paramecium cells served as a test object for the usefulness of conjugation products and optimized assay conditions for EM immunocytochemistry.